US 2015O106974A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0106974 A1 FRANKARD et al. (43) Pub. Date: Apr. 16, 2015

(54) PLANTS HAVING ENHANCED Publication Classification YELD-RELATED TRAITS AND AMETHOD FOR MAKING THE SAME (51) Int. Cl. CI2N 5/82 (2006.01) (71) Applicant: BASF PLANT SCIENCE COMPANY (52) U.S. Cl. GMBH, LUDWIGSHAFEN (DE) CPC ...... CI2N 15/8261 (2013.01); C12N 15/8216 (2013.01) (72) Inventors: VALERIE FRANKARD, WATERLOO (BE); ANDRY ANDRIANKAJA, GENT (BE); YVES HATZFELD, (57) ABSTRACT LILLE (FR), MARIEKE LOUWERS, GENT (BE); STEVEN VANDENABEELE, OUDENAARDE The present invention concerns a method for enhancingyield (BE), AURINEVERKEST, GENT related traits in plants by modulating expression in a plant of (BE); GEERT DE JAEGER, one ore more nucleic acid(s) encoding at least two iSYT (interactor of SYT synovial sarcoma translocation—) EVERGEM (BE): DIRKINZE, polypeptides. The present invention also concerns plants hav MOORSEL-AALST (BE) ing modulated expression of a nucleic acid encoding at least (21) Appl. No.: 14/581,334 two iSYT polypeptides, which plants have enhanced yield related traits relative to corresponding wild type plants or (22) Filed: Dec. 23, 2014 other control plants. Nucleic acids encoding at least two iSYT polypeptides and constructs comprising the same useful in Related U.S. Application Data performing the methods of the invention are also disclosed. Also provided are constructs useful in the methods of the (62) Division of application No. 13/060,881, filed on Apr. invention. The present invention also relates to an iSYT-based 27, 2011, now Pat. No. 8.946,512, filed as application protein complex. The use of the complex to promote plant No. PCT/EP2009/061226 on Aug. 31, 2009. growth, and a method for stimulating the complex formation, (60) Provisional application No. 61/190,543, filed on Aug. by overexpressing at least two members of the complex, are 29, 2008. also disclosed. Patent Application Publication Apr. 16, 2015 Sheet 1 of 3 US 2015/0106974 A1

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PLANTS HAVING ENHANCED achieved. Biomass production is a multi-factorial system in YELD-RELATED TRAITS AND AMETHOD which a plethora of processes are fed into the activity of FOR MAKING THE SAME meristems that give rise to new cells, tissues, and organs. Although a considerable amount of research on yield perfor RELATED APPLICATIONS mance is being performed little is known about the molecular networks underpinning yield (Van Camp, 2005). Many genes 0001. This application is a divisional of patent application have been described in Arabidopsis thaliana that, when Ser. No. 13/060,881 filed Feb. 25, 2011, which is a national mutated or ectopically expressed, result in the formation of stage application (under 35 U.S.C. S371) of PCT/EP2009/ larger structures. Such as leaves or roots. These so-called 061226, filed Aug. 31, 2009, which claims benefit of U.S. “intrinsic yield genes' are involved in many different pro Provisional Application 61/190,543, filed Aug. 29, 2008. The cesses whose interrelationship is mostly unknown. entire content of each aforementioned application is hereby 0006. A trait of particular economic interest is increased incorporated by reference in its entirety. yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of SUBMISSION OF SEQUENCE LISTING quantity and/or quality. Yield is directly dependent on several 0002 The Sequence Listing associated with this applica factors, for example, the number and size of the organs, plant tion is filed in electronic format via EFS-Web and hereby architecture (for example, the number of branches), seed incorporated by reference into the specification in its entirety. production, leaf Senescence and more. Root development, The name of the text file containing the Sequence Listing is nutrient uptake, stress tolerance and early vigour may also be Sequence Listing 074053 001 1 01. The size of the text important factors in determining yield. Optimizing the above file is 2.031 KB, and the text file was created on Dec. 23, 2014. mentioned factors may therefore contribute to increasing 0003. The present invention relates generally to the field of crop yield. molecular biology and concerns a method for enhancing 0007. Seed yield is a particularly important trait, since the yield-related traits in plants by modulating expression in a seeds of many plants are important for human and animal plant of one ore more nucleic acid(s) encoding at least two nutrition. Crops such as corn, rice, wheat, canola and Soybean iSYT (interactor of SYT synovial sarcoma transloca account for over half the total human caloric intake, whether tion—) polypeptides. The present invention also concerns through direct consumption of the seeds themselves or plants having modulated expression of a nucleic acid encod through consumption of meat products raised on processed ing at least two iSYT polypeptides, which plants have seeds. They are also a source of sugars, oils and many kinds of enhanced yield-related traits relative to corresponding wild metabolites used in industrial processes. Seeds contain an type plants or other control plants. The Invention also pro embryo (the Source of new shoots and roots) and an vides hitherto unknown nucleic acids encoding at least two endosperm (the source of nutrients for embryo growth during iSYT polypeptides, and constructs comprising the same, use germination and during early growth of seedlings). The ful in performing the methods of the invention. The invention development of a seed involves many genes, and requires the also provides constructs useful in the methods of the inven transfer of metabolites from the roots, leaves and stems into tion. Furthermore the present invention also relates to an the growing seed. The endosperm, in particular, assimilates iSYT-based protein complex. It further relates to the use of the metabolic precursors of carbohydrates, oils and proteins the complex to promote plant growth, and to a method for and synthesizes them into storage macromolecules to fill out stimulating the complex formation, by overexpressing at least the grain. two members of the complex. 0008 Another important trait for many crops is early 0004. The ever-increasing world population and the dwin vigour. Improving early vigour is an important objective of dling supply of arable land available for agriculture fuels modern rice breeding programs in both temperate and tropi research towards increasing the efficiency of agriculture. cal rice cultivars. Long roots are important for proper soil Conventional means for crop and horticultural improvements anchorage in water-seeded rice. Where rice is sown directly utilise selective breeding techniques to identify plants having into flooded fields, and where plants must emerge rapidly desirable characteristics. However, such selective breeding through water, longer shoots are associated with vigour. techniques have several drawbacks, namely that these tech Where drill-seeding is practiced, longer mesocotyls and niques are typically labour intensive and result in plants that coleoptiles are important for good seedling emergence. The often contain heterogeneous genetic components that may ability to engineer early vigour into plants would be of great not always result in the desirable trait being passed on from importance in agriculture. For example, poor early vigour has parent plants. Advances in molecular biology have allowed been a limitation to the introduction of maize (Zea mays L.) mankind to modify the germplasm of animals and plants. hybrids based on Corn Belt germplasm in the European Genetic engineering of plants entails the isolation and Atlantic. manipulation of genetic material (typically in the form of 0009. A further important trait is that of improved abiotic DNA or RNA) and the subsequent introduction of that genetic stress tolerance. Abiotic stress is a primary cause of crop loss material into a plant. Such technology has the capacity to worldwide, reducing average yields for most major crop deliver crops or plants having various improved economic, plants by more than 50% (Wang et al., Planta 218, 1-14, agronomic or horticultural traits. 2003). Abiotic stresses may be caused by drought, salinity, 0005. The demand for more plant derived products has extremes of temperature, chemical toxicity and oxidative spectacularly increased. In the near future the challenge for stress. The ability to improve plant tolerance to abiotic stress agriculture will be to fulfill the growing demands for feed and would be of great economic advantage to farmers worldwide food in a Sustainable manner. Moreoverplants start to play an and would allow for the cultivation of crops during adverse important role as energy sources. To cope with these major conditions and in territories where cultivation of crops may challenges, a profound increase in plant yield will have to be not otherwise be possible. US 2015/0106974 A1 Apr. 16, 2015

0010 Crop yield may therefore be increased by optimis plant growth (iSYT). Surprisingly, we isolated several pro ing one of the above-mentioned factors. teins belonging to multiprotein complexes. Moreover, many 0011 Depending on the end use, the modification of cer interactors were previously completely uncharacterized. tain yield traits may be favoured over others. For example for Reports on few of the SYT interactors show that they are applications such as forage or wood production, or bio-fuel implicated in several developmental processes (Wagner & resource, an increase in the vegetative parts of a plant may be Meyerowitz, 2002; Meagher et al., 2005; Samowski et al., desirable, and for applications such as flour, starch or oil 2005; Hurtado et al., 2006; Kwon et al., 2006) but so far none production, an increase in seed parameters may be particu of the identified (iSYT genes have been associated with larly desirable. Even amongst the seed parameters, some may stimulation of plant growth. Further Surprising no specific be favoured over others, depending on the application. Vari combination of iSYT polypeptides useful to enhance yield ous mechanisms may contribute to increasing seed yield, related traits has previously been described. whether that is in the form of increased seed size or increased seed number. SUMMARY 0012. One approach to increasing yield (seed yield and/or biomass) in plants may be through modification of the inher 0016 Surprisingly, it has now been found that modulating ent growth mechanisms of a plant, such as the cell cycle or expression in a plant of one ore more nucleic acid(s) encoding various signalling pathways involved in plant growth or in at least two iSYT polypeptides wherein said iSYT polypep defense mechanisms. tide is selected from the group consisting of any of the 0013. It has now been found that various yield-related polypeptides of Table A, homologues thereof, and fusions of traits may be enhanced in plants by modulating expression in the same, promotes plant growth and gives plants having a plant of one or more nucleic acid(s) encoding at least two enhanced yield-related traits relative to control plants. iSYT polypeptides, selected from the group consisting of any 0017. According one embodiment, there is provided a of the polypeptides of Table A, homologues thereof, and method for enhancing yield-related traits in plants relative to fusions of the same. control plants, comprising modulating expression in a plant 0014. One of the abovementioned “intrinsic yield genes', of one or more nucleic acid(s) encoding at least two iSYT AN3 (also known as GIF1 and herein also refer to as SYT polypeptides wherein said iSYT polypeptide is selected from synovial sarcoma translocation polypeptide), was identified the group consisting of any of the polypeptides of Table A, in search of GRF (growth regulating factor) interactors (Kim homologues thereof, and fusions of the same. and Kende, 2004) and by analysis of narrow-leaf Arabidopsis mutants (Horiguchi et al., 2005). SYT is a homolog of the DEFINITIONS human SYT (synovial sarcoma translocation) protein and is encoded by a small gene family in the Arabidopsis genome. Polypeptide(s)/Protein(s) SYT is a transcription co-activator whose biological function, despite the implication of its chromosomal translocation in 0018. The terms “polypeptide' and “protein’ are used tumorigenesis, is still unclear (Clark et al., 1994; de Bruinet interchangeably herein and refer to amino acids in a poly al., 1996). Using the yeast GAL4 system, SYT was shown to meric form of any length, linked together by peptide bonds. possess transactivation activity (Kim and Kende, 2004). This together with yeast two-hybrid and in vitro binding assays Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid demonstrating interaction of SYT with several GRFs (Kim Sequence(s)/Nucleotide Sequence(s) and Kende, 2004; Horiguchi et al., 2005), suggests a role of (0019. The terms “polynucleotide(s)”, “nucleic acid SYT as transcription co-activator of GRFs. GRF (growth sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)'. regulating factor) genes occur in the genomes of all seed “nucleic acid molecule' are used interchangeably herein and plants thus far examined and encode putative transcription refer to nucleotides, either ribonucleotides or deoxyribo factors that play a regulatory role in growth and development nucleotides or a combination of both, in a polymeric of leaves (Kim et al., 2003). In support of a GRF and SYT unbranched form of any length. transcription activator and co-activator complex, grfand SYT mutants display similar phenotypes, and combinations of grf and SYT mutations showed a cooperative effect (Kim and Recombinant DNA Kende, 2004). The SYT mutant narrow-leaf phenotype is 0020 “Recombinant DNA means a DNA molecule that shown to result of a reduction in cell numbers. Moreover, is made by combination of two otherwise separated segments ectopic expression of SYT resulted in transgenic plants with of DNA, e.g., by chemical synthesis or by the manipulation of larger leaves consisting of more cells, indicating that SYT isolated segments of nucleic acids by genetic engineering controls both cell number and organ size (Horiguchi et al., techniques. Recombinant DNA can include exogenous DNA 2005). Although the function of SYT in plant growth regula or simply a manipulated native DNA. Recombinant DNA for tion is not known, these results show that SYT fulfills the expressing a protein in a plant is typically provided as an requirements of an “intrinsic yield gene'. expression cassette which has a promoter that is active in 0015. In our ambition to decipher the molecular network plant cells operably linked to DNA encoding a protein of underpinning yield enhancement mechanism a genome-wide interest. protein centred approach was undertaken to study SYT inter acting proteins in Arabidopsis thaliana cell Suspension cul Homologue(s) tures. The tandem affinity purification (TAP) technology combined with mass spectrometry (MS) based protein iden 0021 “Homologues' of a protein encompass peptides, tification resulted in the isolation and identification of SYT oligopeptides, polypeptides, proteins and enzymes having interacting proteins that may function in the regulation of amino acid Substitutions, deletions and/or insertions relative US 2015/0106974 A1 Apr. 16, 2015 to the unmodified protein in question and having similar Site Directed mutagenesis (Stratagene, San Diego, Calif.), biological and functional activity as the unmodified protein PCR-mediated site-directed mutagenesis or other site-di from which they are derived. rected mutagenesis protocols. 0022. A deletion refers to removal of one or more amino acids from a protein. Derivatives 0023. An insertion refers to one or more amino acid resi dues being introduced into a predetermined site in a protein. 0026 “Derivatives” include peptides, oligopeptides, Insertions may comprise N-terminal and/or C-terminal polypeptides which may, compared to the amino acid fusions as well as intra-sequence insertions of single or mul sequence of the naturally-occurring form of the protein, Such tiple amino adds. Generally, insertions within the amino acid as the protein of interest, comprise Substitutions of amino sequence will be smaller than N- or C-terminal fusions, of the acids with non-naturally occurring amino acid residues, or order of about 1 to 10 residues. Examples of N- or C-terminal additions of non-naturally occurring amino acid residues. fusion proteins or peptides include the binding domain or “Derivatives' of a protein also encompass peptides, oligopep activation domain of a transcriptional activator as used in the tides, polypeptides which comprise naturally occurring yeast two-hybrid system, phage coat proteins, (histidine)-6- altered (glycosylated, acylated, prenylated, phosphorylated, tag, glutathione S-transferase-tag, protein A, maltose-binding myristoylated, Sulphated etc.) or non-naturally altered amino protein, dihydrofolate reductase, Tag 100 epitope, c-myc acid residues compared to the amino acid sequence of a epitope, FLAG-epitope, lacZ, CMP (calmodulin-binding naturally-occurring form of the polypeptide. A derivative peptide), HA epitope, protein C epitope and VSV epitope. may also comprise one or more non-amino acid Substituents 0024. A substitution refers to replacement of amino acids or additions compared to the amino acid sequence from of the protein with other amino acids having similar proper which it is derived, for example a reporter molecule or other ties (such as similar hydrophobicity, hydrophilicity, antige ligand, covalently or non-covalently bound to the amino acid nicity, propensity to form or break C-helical structures or sequence, such as a reporter molecule which is bound to B-sheet structures). Amino add substitutions are typically of facilitate its detection, and non-naturally occurring amino single residues, but may be clustered depending upon func acid residues relative to the amino acid sequence of a natu tional constraints placed upon the polypeptide; insertions will rally-occurring protein. Furthermore, "derivatives” also usually be of the order of about 1 to 10 amino acid residues. include fusions of the naturally-occurring form of the protein The amino acid substitutions are preferably conservative with tagging peptides such as FLAG, HIS6 or thioredoxin (for amino acid substitutions. Conservative substitution tables are a review of tagging peptides, see Terpe, Appl. Microbiol. well known in the art (see for example Creighton (1984) Biotechnol. 60, 523-533, 2003). Proteins. W.H. Freeman and Company (Eds) and Table 1 Orthologue(s)/Paralogue(s) below). 0027 Orthologues and paralogues encompass evolution TABLE 1. ary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have Examples of conserved amino acid substitutions originated through duplication of an ancestral gene; ortho Residue Conservative Substitutions logues are genes from different organisms that have origi nated through speculation, and are also derived from a com Ala Ser Arg Lys mon ancestral gene. ASn Gln: His Asp Glu Domain, Motif/Consensus Sequence/Signature Gln ASn Cys Ser 0028. The term “domain” refers to a set of amino acids Glu Asp conserved at specific positions along an alignment of Gly Pro sequences of evolutionarily related proteins. While amino His ASn; Glin Ile Leu, Val acids at other positions can vary between homologues, amino Leu Ile: Val acids that are highly conserved at specific positions indicate Lys Arg: Gln amino acids that are likely essential in the structure, stability Met Leu: Ile or function of a protein. Identified by their high degree of Phe Met; Leu: Tyr Ser Thr; Gly conservation in aligned sequences of a family of protein Thr Ser; Val homologues, they can be used as identifiers to determine if Trp Tyr any polypeptide in question belongs to a previously identified Tyr Trp; Phe polypeptide family. Wal Ile: Leu 0029. The term “motif or “consensus sequence' or “sig nature” refers to a short conserved region in the sequence of 0.025 Amino acid substitutions, deletions and/or inser evolutionarily related proteins. Motifs are frequently highly tions may readily be made using peptide synthetic techniques conserved parts of domains, but may also include only part of well known in the art, such as Solid phase peptide synthesis the domain, or be located outside of conserved domain (if all and the like, or by recombinant DNA manipulation. Methods of the amino acids of the motif fall outside of a defined for the manipulation of DNA sequences to produce substitu domain). tion, insertion or deletion variants of a protein are well known 0030 Specialist databases exist for the identification of in the art. For example, techniques for making Substitution domains, for example, SMART (Schultz et al. (1998) Proc. mutations at predetermined sites in DNA are well known to Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) those skilled in the art and include M13 mutagenesis, T7-Gen Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and US 2015/0106974 A1 Apr. 16, 2015

Bairoch (1994). A generalized profile syntax for biomolecu query sequence amongst the highest hits; an orthologue is lar sequences motifs and its function in automatic sequence identified if a high-ranking hit in the first BLAST is not from interpretation. (In) ISMB-94: Proceedings 2nd International the same species as from which the query sequence is derived, Conference on Intelligent Systems for Molecular Biology. and preferably results upon BLAST back in the query Altman R., Brutlag D. Karp P. Lathrop R., Searls D., Eds., pp sequence being among the highest hits. 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. 0033 High-ranking hits are those having a low E-value. Res. 32: D134-D137, (2004)), or Pfam (Bateman et al., The lower the E-value, the more significant the score (or in Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools other words the lower the chance that the hit was found by for in silico analysis of protein sequences is available on the chance). Computation of the E-value is well known in the art. ExPASy proteomics server (Swiss Institute of Bioinformatics In addition to E-values, comparisons are also scored by per (Gasteiger et al., ExPASy: the proteomics server for in-depth centage identity. Percentage identity refers to the number of protein knowledge and analysis, Nucleic Acids Res. 31:3784 identical nucleotides (or amino acids) between the two com 3788(2003)). Domains or motifs may also be Identified using pared nucleic acid (or polypeptide) sequences over a particu routine techniques, such as by sequence alignment. lar length. In the case of large families, ClustalW may be 0031 Methods for the alignment of sequences for com used, followed by a neighbour joining tree, to help visualize parison are well known in the art, such methods include GAP, clustering of related genes and to identify orthologues and BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algo paralogues. rithm of Needleman and Wunsch (1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete Hybridisation sequences) alignment of two sequences that maximizes the 0034. The term “hybridisation” as defined herein is a pro number of matches and minimizes the number of gaps. The cess wherein Substantially homologous complementary BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: nucleotide sequences anneal to each other. The hybridisation 403-10) calculates percent sequence identity and performs a process can occur entirely in Solution, i.e. both complemen statistical analysis of the similarity between the two tary nucleic acids are in solution. The hybridisation process sequences. The software for performing BLAST analysis is can also occur with one of the complementary nucleic acids publicly available through the National Centre for Biotech immobilised to a matrix Such as magnetic beads, Sepharose nology Information (NCBI). Homologues may readily be beads or any other resin. The hybridisation process can fur identified using, for example, the ClustalW multiple thermore occur with one of the complementary nucleic acids sequence alignment algorithm (version 1.83), with the default immobilised to a solid support such as a nitro-cellulose or pairwise alignment parameters, and a scoring method in per nylon membrane or immobilised by e.g. photolithography to, centage. Global percentages of similarity and identity may for example, a siliceous glass Support (the latter known as also be determined using one of the methods available in the nucleic acid arrays or microarrays or as nucleic acid chips). In MatCAT software package (Campanella et al., BMC Bioin order to allow hybridisation to occur, the nucleic acid mol formatics. 2003 Jul. 10; 4:29. MatGAT: an application that ecules are generally thermally or chemically denatured to generates similarity/identity matrices using protein or DNA melta double strand into two single Strands and/or to remove sequences.). Minor manual editing may be performed to opti hairpins or other secondary structures from single stranded mise alignment between conserved motifs, as would be nucleic acids. apparent to a person skilled in the art. Furthermore, instead of 0035. The term “stringency” refers to the conditions under using full-length sequences for the identification of homo which a hybridisation takes place. The stringency of hybridi logues, specific domains may also be used. The sequence sation is influenced by conditions such as temperature, salt identity values may be determined over the entire nucleic acid concentration, ionic strength and hybridisation buffer com or amino acid sequence or over selected domains or con position. Generally, low stringency conditions are selected to served motifs), using the programs mentioned above using be about 30°C. lower than the thermal melting point (T) for the default parameters. For local alignments, the Smith-Wa the specific sequence at a defined ionic strength and pH. terman algorithm is particularly useful (Smith TF, Waterman Medium stringency conditions are when the temperature is MS (1981) J. Mol. Biol 147(1):195-7). 20° C. below T. and high stringency conditions are when the temperature is 10° C. below T. High stringency hybridisa Reciprocal BLAST tion conditions are typically used for isolating hybridising 0032 Typically, this involves a first BLAST involving sequences that have high sequence similarity to the target BLASTing a query sequence (for example using any of the nucleic acid sequence. However, nucleic acids may deviate in sequences listed in Table A of the Examples section) against sequence and still encode a Substantially identical polypep any sequence database, such as the publicly available NCBI tide, due to the degeneracy of the genetic code. Therefore database. BLASTN or TBLASTX (using standard default medium stringency hybridisation conditions may sometimes values) are generally used when starting from a nucleotide be needed to identify such nucleic acid molecules. sequence, and BLASTP or TBLASTN (using standard 0036. The Tm is the temperature under defined ionic default values) when starting from a protein sequence. The strength and pH, at which 50% of the target sequence hybri BLAST results may optionally be filtered. The full-length dises to a perfectly matched probe. The T is dependent upon sequences of either the filtered results or non-filtered results the solution conditions and the base composition and length are then BLASTedback (second BLAST) against sequences of the probe. For example, longer sequences hybridise spe from the organism from which the query sequence is derived. cifically at higher temperatures. The maximum rate of The results of the first and second BLASTs are then com hybridisation is obtained from about 16° C. up to 32° C. pared. A paralogue is identified if a high-ranking hit from the below T. The presence of monovalent cations in the hybridi first blast is from the same species as from which the query sation solution reduce the electrostatic repulsion between the sequence is derived, a BLAST back then ideally results in the two nucleic acid strands thereby promoting hybrid formation; US 2015/0106974 A1 Apr. 16, 2015

this effect is visible for sodium concentrations of up to 0.4M 1xSSC and 50% formamide, followed by washing at 65°C. in (for higher concentrations, this effect may be ignored). For 0.3xSSC. Examples of medium stringency hybridisation con mamide reduces the melting temperature of DNA-DNA and ditions for DNA hybrids longer than 50 nucleotides encom DNA-RNA duplexes with 0.6 to 0.7° C. for each percent pass hybridisation at 50° C. in 4xSSC or at 40°C. in 6xSSC formamide, and addition of 50% formamide allows hybridi and 50% formamide, followed by washing at 50° C. in sation to be performed at 30 to 45° C., though the rate of 2xSSC. The length of the hybrid is the anticipated length for hybridisation will be lowered. Base pair mismatches reduce the hybridising nucleic acid. When nucleic acids of known the hybridisation rate and the thermal stability of the sequence are hybridised, the hybrid length may be deter duplexes. On average and for large probes, the Tm decreases mined by aligning the sequences and identifying the con about 1° C. per% base mismatch. The Tm may be calculated served regions described herein. 1xSSC is 0.15M NaCl and using the following equations, depending on the types of 15 mM sodium citrate; the hybridisation solution and wash hybrids: Solutions may additionally include 5xDenhardt's reagent, 1) DNA-DNA Hybrids (Meinkoth and Wahl, Anal. Biochem. 0.5-1.0% SDS, 100 ug/mil denatured, fragmented salmon 138: 267-284, 1984): sperm DNA, 0.5% sodium pyrophosphate. T=81.5° C.+16.6xlogo Na+0.41x%G/C-500x 0043. For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular L'I'-0.61x% formamide Cloning: a laboratory manual, 3" Edition, Cold Spring Har bor Laboratory Press, CSH, New York or to Current Protocols 2) DNA-RNA or RNA-RNA Hybrids: in Molecular Biology, John Wiley & Sons, N.Y. (1989 and 0037 yearly updates). Splice Variant 3) oligo-DNA or oligo-RNA hybrids: 0044) The term “splice variant as used herein encom 0038. For <20 nucleotides: T-2 (I) passes variants of a nucleic acid sequence in which selected 0039. For 20-35 nucleotides: T-22+1.46 (I) introns and/or exons have been excised, replaced, displaced “ or for other monovalent cation, but only accurate in the or added, or in which introns have been shortened or length 0.01-0.4M range. ened. Such variants will be ones in which the biological only accurate for % GC in the 30% to 75% range. activity of the protein is substantially retained; this may be L=length of duplex in base pairs. achieved by selectively retaining functional segments of the "oligo, oligonucleotide; I, effective length of primer-2x(no. protein. Such splice variants may be found in nature or may be of G/C)+(no. of A/T). manmade. Methods for predicting and isolating Such splice 0040. Non-specific binding may be controlled using any variants are well known in the art (see for example Foissac one of a number of known techniques such as, for example, and Schiex (2005) BMC Bioinformatics 6:25). blocking the membrane with protein containing Solutions, additions of heterologous RNA, DNA, and SDS to the Allelic Variant hybridisation buffer, and treatment with Rnase. For non-ho 0045 Alleles or allelic variants are alternative forms of a mologous probes, a series of hybridizations may be per given gene, located at the same chromosomal position. Allelic formed by varying one of (i) progressively lowering the variants encompass Single Nucleotide Polymorphisms annealing temperature (for example from 68°C. to 42°C.) or (SNPs), as well as Small Insertion/Deletion Polymorphisms (ii) progressively lowering the formamide concentration (for (INDELs). The size of INDELs is usually less than 100 bp. example from 50% to 0%). The skilled artisan is aware of SNPs and INDELS form the largest set of sequence variants in various parameters which may be altered during hybridisa naturally occurring polymorphic strains of most organisms. tion and which will either maintain or change the stringency conditions. Endogenous Gene 0041 Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post 0046 Reference herein to an “endogenous gene not only hybridisation washes. To remove background resulting from refers to the gene in question as found in a plant in its natural non-specific hybridisation, Samples are washed with dilute form (i.e., without there being any human intervention), but salt solutions. Critical factors of such washes include the also refers to that same gene (or a substantially homologous ionic strength and temperature of the final wash solution: the nucleic acid/gene) in an isolated form Subsequently (re)intro lower the salt concentration and the higher the wash tempera duced into a plant (a transgene). For example, a transgenic ture, the higher the stringency of the wash. Wash conditions plant containing Such a transgene may encountera Substantial are typically performed at or below hybridisation Stringency. reduction of the transgene expression and/or substantial A positive hybridisation gives a signal that is at least twice of reduction of expression of the endogenous gene. The isolated that of the background. Generally, Suitable stringent condi gene may be isolated from an organism or may be manmade, tions for nucleic acid hybridisation assays or gene amplifica for example by chemical synthesis. tion detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan Gene Shuffling/Directed Evolution is aware of various parameters which may be altered during 0047 Gene shuffling or directed evolution consists of washing and which will either maintain or change the strin iterations of DNA shuffling followed by appropriate screen gency conditions. ing and/or selection to generate variants of nucleic acids or 0042. For example, typical high stringency hybridisation portions thereof encoding proteins having a modified biologi conditions for DNA hybrids longer than 50 nucleotides cal activity (Castle et al., (2004) Science 304(5674): 1151-4: encompass hybridisation at 65° C. in 1xSSC or at 42°C. in U.S. Pat. Nos. 5,811.238 and 6,395,547). US 2015/0106974 A1 Apr. 16, 2015

Construct “plant promoter can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid 0048. Additional regulatory elements may include tran sequence to be expressed in the inventive process and Scriptional as well as translational enhancers. Those skilled in described herein. This also applies to other “plant” regulatory the art will be aware of terminator and enhancer sequences signals, such as “plant terminators. The promoters upstream that may be suitable for use in performing the invention. An of the nucleotide sequences useful in the methods of the intron sequence may also be added to the 5' untranslated present invention can be modified by one or more nucleotide region (UTR) or in the coding sequence to increase the substitution(s), insertion(s) and/or deletion(s) without inter amount of the mature message that accumulates in the cyto fering with the functionality or activity of either the promot sol, as described in the definitions section. Other control ers, the open reading frame (ORF) or the 3'-regulatory region sequences (besides promoter, enhancer, silencer, intron Such as terminators or other 3' regulatory regions which are sequences, 3'UTR and/or 5' UTR regions) may be protein located away from the ORF. It is furthermore possible that the and/or RNA stabilizing elements. Such sequences would be activity of the promoters is increased by modification of their known or may readily be obtained by a person skilled in the sequence, or that they are replaced completely by more active art promoters, even promoters from heterologous organisms. For 0049. The genetic constructs of the invention may further expression in plants, the nucleic acid molecule must, as include an origin of replication sequence that is required for described above, be linked operably to or comprise a suitable maintenance and/or replication in a specific cell type. One promoter which expresses the gene at the right point in time example is when a genetic construct is required to be main and with the required spatial expression pattern. tained in a bacterial cell as an episomal genetic element (e.g. 0053 For the identification of functionally equivalent pro plasmid or cosmid molecule). Preferred origins of replication moters, the promoter strength and/or expression pattern of a include, but are not limited to, the fl-ori and colE1. candidate promoter may be analysed for example by operably 0050 For the detection of the successful transfer of the linking the promoter to a reporter gene and assaying the nucleic acid sequences as used in the methods of the invention expression level and pattern of the reporter gene in various and/or selection of transgenic plants comprising these nucleic tissues of the plant. Suitable well-known reporter genes acids, it is advantageous to use marker genes (or reporter include for example beta-glucuronidase or beta-galactosi genes). Therefore, the genetic construct may optionally com dase. The promoteractivity is assayed by measuring the enzy prise a selectable marker gene. Selectable markers are matic activity of the beta-glucuronidase or beta-galactosi described in more detail in the "definitions' section herein. dase. The promoter strength and/or expression pattern may The marker genes may be removed or excised from the trans then be compared to that of a reference promoter (such as the genic cell once they are no longer needed. Techniques for one used in the methods of the present invention). Alterna marker removal are known in the art, useful techniques are tively, promoter strength may be assayed by quantifying described above in the definitions section. mRNA levels or by comparing mRNA levels of the nucleic acid used in the methods of the present invention, with mRNA Regulatory Element/Control Sequence/Promoter levels of housekeeping genes such as 18S rRNA, using meth 0051. The terms “regulatory element”, “control sequence' ods known in the art, such as Northern blotting with densito and “promoter are all used interchangeably herein and are to metric analysis of autoradiograms, quantitative real-time be taken in a broad context to refer to regulatory nucleic acid PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: sequences capable of effecting expression of the sequences to 986-994). Generally by “weak promoter is intended a pro which they are ligated. The term “promoter typically refers moter that drives expression of a coding sequence at a low to a nucleic acid control sequence located upstream from the level. By “low level” is intended at levels of about 1/10,000 transcriptional start of a gene and which is involved in recog transcripts to about 11 100,000 transcripts, to about 1/500, nising and binding of RNA polymerase and other proteins, 0000 transcripts per cell. Conversely, a “strong promoter” thereby directing transcription of an operably linked nucleic drives expression of a coding sequence at high level, or at acid. Encompassed by the aforementioned terms are tran about 1/10 transcripts to about 1/100 transcripts to about Scriptional regulatory sequences derived from a classical 1/1000 transcripts per cell. Generally, by “medium strength eukaryotic genomic gene (including the TATA box which is promoter is intended a promoter that drives expression of a required for accurate transcription initiation, with or without coding sequence at a lower level than a strong promoter, in a CCAAT box sequence) and additional regulatory elements particular at a level that is in all instances below that obtained (i.e. upstream activating sequences, enhancers and silencers) when under the control of a 35S CaMV promoter. which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also Operably Linked included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may 0054) The term “operably linked” as used herein refers to include a 35 box sequence and/or—10 box transcriptional a functional linkage between the promoter sequence and the regulatory sequences. The term “regulatory element also gene of interest, such that the promoter sequence is able to encompasses a synthetic fusion molecule or derivative that initiate transcription of the gene of interest. confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ. Constitutive Promoter 0052 A “plant promoter comprises regulatory elements, 0055. A "constitutive promoter refers to a promoter that which mediate the expression of a coding sequence segment is transcriptionally active during most, but not necessarily all, in plant cells. Accordingly, a plant promoter need not be of phases of growth and development and under most environ plant origin, but may originate from viruses or micro-organ mental conditions, in at least one cell, tissue or organ. Table 2a isms, for example from viruses which attack plant cells. The below gives examples of constitutive promoters. US 2015/0106974 A1 Apr. 16, 2015

TABLE 2a whilst still allowing for any leaky expression in these other plant parts. Promoters able to initiate transcription in certain Examples of constitutive promoters cells only are referred to herein as “cell-specific'. Gene Source Reference 0060 Examples of root-specific promoters are listed in Actin McElroy et al, Plant Cell, 2:163–171, 1990 Table 2b below: HMGP WO 2004,070039 CAMV 3SS Odell etal, Nature, 313: 810-812, 1985 TABLE 2b CaMV 19S Nilsson et al., Physiol. Plant. 100:456-462, 1997 GOS2 de Pater et al, Plant J Nov; 2(6): 837-44, 1992, WO 2004,065596 Examples of root-specific promoters Ubiquitin Christensen etal, Plant Mol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al., Plant Mol Biol. 25(5): 837-43, 1994 Gene Source Reference Maize H3 histone Lepetit etal, Mol. Gen. Genet. 231: 276-285, 1992 Alfalfa H3 Wu et al. Plant Mol. Biol. 11: 641-649, 1988 RCc3 Plant Mol Biol. 1995 January; 27(2): 237-48 histone Arabidopsis PHT1 Kovama et al., 2005; Mudge et al. (2002, Actin 2 An etal, Plant J. 10(1): 107-121, 1996 Plant J. 31:341) 34S FMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Medicago phosphate Xiao et al., 2006 Rubisco small U.S. Pat. No. 4,962,028 Subunit transporter OCS Leisner (1988) Proc Natl AcadSci USA 85(5): 2553 Arabidopsis Pyk10 Nitz et al. (2001) Plant Sci 161(2): 337-346 SAD1 Jain et al., CropScience, 39 (6), 1999: 1696 root-expressible genes Tingey et al., EMBO J. 6: 1, 1987. SAD2 Jain et al., CropScience, 39 (6), 1999: 1696 tobacco auxin Van der Zaal et al., Plant Mol. Biol. 16, OS Shaw et al. (1984) Nucleic Acids Res. 12(20): inducible gene 983, 1991. 7831-7846 B-tubulin Oppenheimer, et al., Gene 63: 87, 1988. V-ATPase WOO1,14572 tobacco root Conkling, et al., Plant Physiol. 93: 1203, 1990. Super promoter WO95/14098 specific genes G-box proteins WO 94f12015 B. naptis G1-3b gene U.S. Pat. No. 5,401,836 SbPRP1 Suzuki et al., Plant Mol. Biol. 21: 109-119, 993. Ubiquitous Promoter LRX1 Baumberger et al. 2001, Genes & Dev. 15: 1128 BTG-26 Brassica US 20OSOO44585 0056. A ubiquitous promoter is active in substantially all naptiS tissues or cells of an organism. LeAMT1 (tomato) Lauter et al. (1996, PNAS 3:8139) The LeNRT1-1 Lauter et al. (1996, PNAS 3: 8,139) Developmentally-Regulated Promoter (tomato) class I patatin Liu et al., Plant Mol. Biol. 153: 386–395, 1991. 0057. A developmentally-regulated promoter is active gene (potato) during certain developmental stages or in parts of the plant KDC1 (Daucus Downey et al. (2000, J. Biol. Chem. 275: 39420) that undergo developmental changes. carota) TobRB7 gene W Song (1997) PhD Thesis, North Carolina State Inducible Promoter University, Raleigh, NC USA OsRAB5a (rice) Wang et al. 2002, Plant Sci. 163: 273 0058 An inducible promoter has induced or increased ALF5 (Arabidopsis) Diener et al. (2001, Plant Cell 13: 1625) transcription initiation in response to a chemical (for a review NRT2; 1Np (N. Quesada et al. (1997, Plant Mol. Biol. 34: 265) see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., plumbaginifolia) 48:89-108), environmental or physical stimulus, or may be “stress-inducible', i.e. activated when a plant is exposed to various stress conditions, or a "pathogen-inducible' i.e. acti 0061 A seed-specific promoter is transcriptionally active vated when a plant is exposed to exposure to various patho predominantly in seed tissue, but not necessarily exclusively genS. in seed tissue (in cases of leaky expression). The seed-specific promoter may be active during seed development and/or dur Organ-Specific/Tissue-Specific Promoter ing germination. The seed specific promoter may be 0059 An organ-specific or tissue-specific promoter is one endosperm/aleurone? embryo specific. Examples of seed-spe that is capable of preferentially initiating transcription in cific promoters (endosperm/aleurone/embryo specific) are certain organs or tissues. Such as the leaves, roots, seed tissue shown in Table 2c to Table 2f below. Further examples of etc. For example, a “root-specific promoter is a promoter seed-specific promoters are given in Qing Qu and Takaiwa that is transcriptionally active predominantly in plant roots, (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure is Substantially to the exclusion of any other parts of a plant, incorporated by reference herein as if fully set forth. TABLE 2c Examples of seed-specific promoters

Gene Source Reference seed-specific genes Simon et al., Plant Mol. Biol. 5: 191, 1985; Scofield et al., J. Biol. Chem. 262: 12202, 1987.: Baszczynski et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin Pearson et al., Plant Mol. Biol. 18: 235-245, 1992. legumin Ellis et al., Plant Mol. Biol. 10:203-214, 1988. US 2015/0106974 A1 Apr. 16, 2015

TABLE 2c-continued Examples of seed-specific promoters

Gene Source Reference glutelin (rice) Takaiwa et al., Mol. Gen. Genet. 208: 15-22, 1986: Takaiwa et al., FEBS Letts. 221:43-47, 1987. Zein Matzke etal Plant Mol Biol, 14(3): 323-32 1990 nap A Stalberg etal, Planta 199: 515-519, 1996. wheat LMW and HMW Mol Gen Genet 216: 81–90, 1989; NAR 17:461-2, 1989 glutenin-1 wheat SPA Albani etal, Plant Cell, 9: 171-184, 1997 wheat Cl, f, Y-gliadins EMBO J. 3: 1409-15, 1984 barley Itrl promoter Diaz et al. (1995) Mol Gen Genet 248(5): 592-8 barley B1, C, D, hordein Theor Appl Gen98: 1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250: 750-60, 1996 barley DOF Mena et al. The Plant Journal, 116(1): 53-62, 1998 blz2 EP991 O6056.7 synthetic promoter Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998. rice prolamin NRP33 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 rice a-globulin Glb-1 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 rice OSH1 Sato et al., Proc. Natl. Acad. Sci. USA, 93:8117-8122, 1996 rice C-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33: 513-522, 1997 rice ADP-glucose pyrophos Trans Res 6: 157-68, 1997 phorylase maize ESR gene family Plant J 12: 235-46, 1997 Sorghiin C-kafirin DeRose et al., Plant Mol. Biol 32: 1029-35, 1996 KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 rice oleosin Wu et al., J. Biochem. 123: 386, 1998 Sunflower oleosin Cummins et al., Plant Mol. Biol. 19: 873-876, 1992 PROO117, putative rice 40S WO 2004,070039 ribosomal protein PROO136, rice alanine unpublished aminotransferase PROO147, trypsin inhibitor unpublished TR1 (barley) PROO151, rice WSI18 WO 2004,070039 PROO175, rice RAB21 WO 2004,070039 PROOO5 WO 2004,070039 PROOO95 WO 2004,070039 C-amylase (Amy32b) Lanahan etal, Plant Cell 4: 203-211, 1992: Skriver etal, Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin B-like gene Cejudo et al, Plant Mol Biol 20:849-856, 1992 Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 Chi26 Leah et al., Plant J. 4:579-89, 1994 Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998

TABLE 2d TABLE 2d-continued examples of endosperm-specific promoters examples of endosperm-specific promoters

Gene Source Reference Gene Source Reference glutelin (rice) Takaiwa et al. (1986) Mol Gen Genet 208: 15-22; rice globulin Nakase et al. (1997) Plant Molec Biol 33: 513-522 Takaiwa et al. (1987) FEBS Letts. 221:43-47 REB, OHP-1 Zein Matzke et al., (1990) Plant Mol Biol 14(3): 323-32 rice ADP-glucose Russell et al. (1997) Trans Res 6: 157-68 wheat LMW Colotet al. (1989) Mol Gen Genet 216: 81-90, pyrophosphorylase and HMW Anderson et al. (1989) NAR 17:461-2 maize ESR Opsahl-Ferstad et al. (1997) Plant J 12: 235-46 glutenin-1 gene family wheat SPA Albani et al. (1997) Plant Cell 9: 171-184 Sorghum kafirin DeRose et al. (1996) Plant Mol Biol 32: 1029-35 wheat gliadins Rafalski et al. (1984) EMBO 3: 1409-15 barley Itrl Diaz et al. (1995) Mol Gen Genet 248 (5): 592-8 promoter barley B1, C, D, Cho et al. (1999) Theor Appl Genet 98: 1253-62: TABLE 2e hordein Muller et al. (1993) Plant J 4:343-55; Sorenson et al. (1996) Mol Gen Genet 250: 750-60 Examples of embryo specific promoters: barley DOF Mena et al., (1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem 274(14):9175-82 Gene Source Reference synthetic promoter Vicente-Carbajosa et al. (1998) Plant J 13: 629-640 rice OSH1 Sato et al., Proc. Natl. Acad. Sci. USA, 93:8117-8122, rice prolamin Wu et al., (1998) Plant Cell Physiol 39(8) 885-889 1996 NRP33 KNOX Postma-Haarsma etal, Plant Mol. Biol. 39: 257-71, 1999 rice globulin Wu et al. (1998) Plant Cell Physiol 39(8) 885-889 PROO151 WO 2004f070039 Gb-1 PROO175 WO 2004f070039 US 2015/0106974 A1 Apr. 16, 2015

TABLE 2e-continued Terminator Examples of embryo specific promoters: 0065. The term “terminator encompasses a control sequence which is a DNA sequence at the end of a transcrip Gene Source Reference tional unit which signals 3' processing and polyadenylation of PROOO5 WO 2004f070039 a primary transcript and termination of transcription. The PROOO95 WO 2004f070039 terminator can be derived from the natural gene, from a vari ety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline Syn TABLE 2f thase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukary Examples of aleurOne-Specific promoters: otic gene. Gene Source Reference Selectable Marker (Gene)/Reporter Gene C-amylase Lanahan etal, Plant Cell 4: 203-211, 1992: (Amy32b) Skriver etal, Proc Natl Acad Sci USA 88: 7266-7270, 1991 0066 “Selectable marker”, “selectable marker gene” or cathepsin B-like gene Cejudo et al, Plant Mol Biol 20:849-856, 1992 “reporter gene' includes any gene that confers a phenotype on Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 a cell in which it is expressed to facilitate the identification Chi26 Leah et al., Plant J. 4: 579-89, 1994 and/or selection of cells that are transfected or transformed Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998 with a nucleic acid construct of the invention. These marker genes enable the identification of a successful transfer of the 0062) Agreen tissue-specific promoter as defined herein is nucleic acid molecules via a series of different principles. a promoter that is transcriptionally active predominantly in Suitable markers may be selected from markers that confer green tissue, Substantially to the exclusion of any other parts antibiotic or herbicide resistance, that introduce a new meta of a plant, whilst still allowing for any leaky expression in bolic trait or that allow visual selection. Examples of select these other plant parts. able marker genes include genes conferring resistance to 0063 Examples of green tissue-specific promoters which antibiotics (such as nptII that phosphorylates neomycin and may be used to perform the methods of the invention are kanamycin, or hpt, phosphorylating hygromycin, or genes shown in Table 2g below. conferring resistance to, for example, bleomycin, streptomy cin, tetracyclin, chloramphenicol, amplicillin, gentamycin, TABLE 2g geneticin (G418), spectinomycin or blasticidin), to herbi Examples of green tissue-specific promoters cides (for example bar which provides resistance to Basta R; aroA or goX providing resistance against glyphosate, or the Gene Expression Reference genes conferring resistance to, for example, imidazolinone, Maize Orthophosphate dikinase Leaf specific Fukavama et al., 2001 phosphinothricin or Sulfonylurea), or genes that provide a Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001 metabolic trait (such as man A that allows plants to use man carboxylase nose as sole carbon source or Xylose isomerase for the utili Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003 carboxylase sation of Xylose, or antinutritive markers such as the resis Rice small subunit Rubisco Leaf specific Nomura et al., 2000 tance to 2-deoxyglucose). Expression of visual marker genes rice beta expansin EXBP9 Shoot specific WO 2004/070039 results in the formation of colour (for example B-glucu Pigeonpea Small subunit Rubisco Leaf specific Panguluri et al., 2005 ronidase, GUS or 3-galactosidase with its coloured sub Pea RBCS3A Leaf specific strates, for example X-Gal), luminescence (such as the luciferin?luceferase system) or fluorescence (Green Fluores 0064. Another example of a tissue-specific promoter is a cent Protein, GFP, and derivatives thereof). This list repre meristem-specific promoter, which is transcriptionally active sents only a small number of possible markers. The skilled predominantly in meristematic tissue, Substantially to the worker is familiar with such markers. Different markers are exclusion of any other parts of a plant, whilst still allowing for preferred, depending on the organism and the selection any leaky expression in these other plant parts. Examples of method. green meristem-specific promoters which may be used to 0067. It is known that upon stable or transient integration perform the methods of the invention are shown in Table 2h of nucleic acids into plant cells, only a minority of the cells below. takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the TABLE 2h transfection technique used. To identify and select these inte Examples of meristen-specific promoters grants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells Gene Source Expression pattern Reference together with the gene of interest. These markers can for rice OSH1 Shoot apical meristem, Sato et al. (1996) example be used in mutants in which these genes are not from embryo globular Proc. Natl. Acad. Sci. functional by, for example, deletion by conventional meth stage to seedling stage USA, 93:8117-8122 ods. Furthermore, nucleic acid molecules encoding a select Rice metallothionein Meristem specific BAD87835.1 able marker can be introduced into a host cell on the same WAK1 & WAK2 Shoot and root apical Wagner & Kohorn meristems, and in ex (2001) Plant Cell vector that comprises the sequence encoding the polypeptides panding leaves and sepals 13(2): 303-318 of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably trans fected with the introduced nucleic acid can be identified for US 2015/0106974 A1 Apr. 16, 2015

example by selection (for example, cells which have inte the modification to take the form of, for example, a substitu grated the selectable marker survive whereas the other cells tion, addition, deletion, inversion or insertion of one or more die). nucleotide residues. The natural genetic environment is 0068. Since the marker genes, particularly genes for resis understood as meaning the natural genomic or chromosomal tance to antibiotics and herbicides, are no longer required or locus in the original plant or the presence in a genomic library. are undesired in the transgenic host cell once the nucleic acids have been introduced Successfully, the process according to In the case of a genomic library, the natural genetic environ the invention for introducing the nucleic acids advanta ment of the nucleic acid sequence is preferably retained, at geously employs techniques which enable the removal or least in part. The environment flanks the nucleic acid excision of these marker genes. One such a method is what is sequence at least on one side and has a sequence length of at known as co-transformation. The co-transformation method least 50 bp, preferably at least 500 bp, especially preferably at employs two vectors simultaneously for the transformation, least 1000 bp, most preferably at least 5000 bp. A naturally one vector bearing the nucleic acid according to the invention occurring expression cassette—for example the naturally and a second bearing the marker gene(s). A large proportion occurring combination of the natural promoter of the nucleic of transformants receives or, in the case of plants, comprises acid sequences with the corresponding nucleic acid sequence (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants encoding a polypeptide useful in the methods of the present usually receive only a part of the vector, i.e. the sequence invention, as defined above becomes a transgenic expres flanked by the T-DNA, which usually represents the expres sion cassette when this expression cassette is modified by sion cassette. The marker genes can Subsequently be removed non-natural, synthetic (“artificial) methods such as, for from the transformed plant by performing crosses. In another example, mutagenic treatment. Suitable methods are method, marker genes integrated into a transposon are used described, for example, in U.S. Pat. No. 5,565,350 or WO for the transformation together with desired nucleic acid OOf 15815. (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are 0073. A transgenic plant for the purposes of the invention transformed with a nucleic acid construct conferring expres is thus understood as meaning, as above, that the nucleic acids sion of a transposase, transiently or stable. In some cases used in the method of the invention are not at their natural (approx. 10%), the transposon jumps out of the genome of the locus in the genome of said plant, it being possible for the host cell once transformation has taken place Successfully nucleic acids to be expressed homologously or heterolo and is lost. In a further number of cases, the transposon jumps gously. However, as mentioned, transgenic also means that, to a different location. In these cases the marker gene must be while the nucleic acids according to the invention or used in eliminated by performing crosses. In microbiology, tech the inventive method are at their natural position in the niques were developed which make possible, or facilitate, the genome of a plant, the sequence has been modified with detection of Such events. A further advantageous method regard to the natural sequence, and/or that the regulatory relies on what is known as recombination systems; whose sequences of the natural sequences have been modified. advantage is that elimination by crossing can be dispensed Transgenic is preferably understood as meaning the expres with. The best-known system of this type is what is known as sion of the nucleic acids according to the invention at an the Creflox system. Cre1 is a recombinase that removes the unnatural locus in the genome, i.e. homologous or, prefer sequences located between the loXP sequences. If the marker ably, heterologous expression of the nucleic acids takes place. gene is integrated between the loXP sequences, it is removed once transformation has taken place Successfully, by expres Preferred transgenic plants are mentioned herein. sion of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., Modulation J. Biol. Chem.,275, 2000: 22255-22267; Velmuruganet al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration 0074 The term “modulation” means in relation to expres into the plant genome of the nucleic acid sequences according sion or gene expression, a process in which the expression to the invention is possible. Naturally, these methods can also level is changed by said gene expression in comparison to the be applied to microorganisms such as yeast, fungi or bacteria. control plant, the expression level may be increased or Transgenic/Transgene/Recombinant decreased. The original, unmodulated expression may be of 0069. For the purposes of the invention, “transgenic’, any kind of expression of a structural RNA (rRNA, tRNA) or “transgene' or “recombinant’ means with regard to, for mRNA with subsequent translation. The term “modulating example, a nucleic acid sequence, an expression cassette, the activity” shall mean any change of the expression of the gene construct or a vector comprising the nucleic acid inventive nucleic acid sequences or encoded proteins, which sequence or an organism transformed with the nucleic acid leads to increased yield and/or increased growth of the plants. sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombi Expression nant methods in which either 0070 (a) the nucleic acid sequences encoding proteins 0075. The term “expression' or “gene expression' means useful in the methods of the invention, or the transcription of a specific gene or specific genes or spe 0071 (b) genetic control sequence(s) which is operably cific genetic construct. The term "expression' or 'gene linked with the nucleic acid sequence according to the expression' in particular means the transcription of a gene or invention, for example a promoter, or genes or genetic construct into structural RNA (rRNA, tRNA) (0072 (c) a) and b) or mRNA with or without subsequent translation of the latter are not located in their natural genetic environment or have into a protein. The process includes transcription of DNA and been modified by recombinant methods, it being possible for processing of the resulting mRNA product. US 2015/0106974 A1 Apr. 16, 2015

Increased Expression/Overexpression tein of interest. Preferably, the stretch of substantially con tiguous nucleotides is capable of forming hydrogen bonds 0076. The term “increased expression' or “overexpres with the target gene (either sense or antisense Strand), more sion” as used herein means any form of expression that is preferably, the stretch of substantially contiguous nucleotides additional to the original wild-type expression level. has, in increasing order of preference, 50%, 60%, 70%, 80%, 0077 Methods for increasing expression of genes or gene 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence products are well documented in the art and include, for identity to the target gene (either sense orantisense strand). A example, overexpression driven by appropriate promoters, nucleic acid sequence encoding a (functional) polypeptide is the use of transcription enhancers or translation enhancers. not a requirement for the various methods discussed herein Isolated nucleic acids which serve as promoter or enhancer for the reduction or substantial elimination of expression of elements may be introduced in an appropriate position (typi an endogenous gene. cally upstream) of a non-heterologous form of a polynucle otide so as to upregulate expression of a nucleic acid encoding I0082. This reduction or substantial elimination of expres the polypeptide of interest. For example, endogenous pro sion may be achieved using routine tools and techniques. A moters may be altered in vivo by mutation, deletion, and/or preferred method for the reduction or substantial elimination substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling at of endogenous gene expression is by introducing and express al., WO9322443), or isolated promoters may be introduced ing in a plant a genetic construct into which the nucleic acid into a plant cell in the proper orientation and distance from a (in this case a stretch of Substantially contiguous nucleotides gene of the present invention so as to control the expression of derived from the gene of interest, or from any nucleic acid the gene. capable of encoding an orthologue, paralogue or homologue 0078 If polypeptide expression is desired, it is generally of any one of the protein of interest) is cloned as an inverted desirable to include a polyadenytation region at the 3'-end of repeat (in part or completely), separated by a spacer (non a polynucleotide coding region. The polyadenylation region coding DNA). can be derived from the natural gene, from a variety of other I0083. In such a preferred method, expression of the endog plant genes, or from T-DNA. The 3' end sequence to be added enous gene is reduced or Substantially eliminated through may be derived from, for example, the nopaline synthase or RNA-mediated silencing using an inverted repeat of a nucleic octopine synthase genes, or alternatively from another plant acid or a part thereof (in this case a stretch of substantially gene, or less preferably from any other eukaryotic gene. contiguous nucleotides derived from the gene of interest, or 0079 An intron sequence may also be added to the 5' from any nucleic acid capable of encoding an orthologue, untranslated region (UTR) or the coding sequence of the paralogue or homologue of the protein of interest), preferably partial coding sequence to increase the amount of the mature capable of forming a hairpin structure. The inverted repeat is message that accumulates in the cytosol. Inclusion of a spilce cloned in an expression vector comprising control sequences. able intron in the transcription unit in both plant and animal A non-coding DNA nucleic acid sequence (a spacer, for expression constructs has been shown to increase gene example a matrix attachment region fragment (MAR), an expression at both the mRNA and protein levels up to 1000 intron, a polylinker, etc.) is located between the two inverted fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405: nucleic acids forming the inverted repeat. After transcription Callis et al. (1987) Genes Dev 1:1 183-1200). Such intron of the inverted repeat, a chimeric RNA with a self-comple enhancement of gene expression is typically greatest when mentary structure is formed (partial or complete). This placed near the 5' end of the transcription unit. Use of the double-stranded RNA structure is referred to as the hairpin maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron RNA (hpRNA). The hpRNA is processed by the plant into are known in the art. For general information see: The Maize siRNAs that are incorporated into an RNA-induced silencing Handbook, Chapter 116, Freeling and Walbot, Eds. Springer, complex (RISC). The RISC further cleaves the mRNA tran N.Y. (1994). scripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides. For furthergen Decreased Expression eral details see for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO99/53050). 0080 Reference herein to “decreased expression” or “reduction or substantial elimination of expression is taken 0084 Performance of the methods of the invention does to mean a decrease in endogenous gene expression and/or not rely on introducing and expressing in a plant a genetic polypeptide levels and/or polypeptide activity relative to con construct into which the nucleic acid is cloned as an inverted trol plants. The reduction or substantial elimination is in repeat, but any one or more of several well-known gene increasing order of preference at least 10%, 20%, 30%, 40% silencing methods may be used to achieve the same effects. or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, I0085. One such method for the reduction of endogenous 98%, 99% or more reduced compared to that of control plants. gene expression is RNA-mediated silencing of gene expres 0081 For the reduction or substantial elimination of sion (downregulation). Silencing in this case is triggered in a expression an endogenous gene in a plant, a sufficient length plant by a double stranded RNA sequence (dsRNA) that is of Substantially contiguous nucleotides of a nucleic acid Substantially similar to the target endogenous gene. This sequence is required. In order to perform gene silencing, this dsRNA is further processed by the plant into about 20 to about may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 26 nucleotides called short interfering RNAs (siRNAs). The fewer nucleotides, alternatively this may be as much as the siRNAs are incorporated into an RNA-induced silencing entire gene (including the 5' and/or 3' UTR, either in part or in complex (RISC) that cleaves the mRNA transcript of the whole). The stretch of substantially contiguous nucleotides endogenous target gene, thereby Substantially reducing the may be derived from the nucleic acid encoding the protein of number of mRNA transcripts to be translated into a polypep interest (target gene), or from any nucleic acid capable of tide. Preferably, the double stranded RNA sequence corre encoding an orthologue, paralogue or homologue of the pro sponds to a target gene. US 2015/0106974 A1 Apr. 16, 2015

I0086. Another example of an RNA silencing method I0089. The antisense nucleic acid sequence can be pro involves the introduction of nucleic acid sequences or parts duced biologically using an expression vector into which a thereof (in this case a stretch of Substantially contiguous nucleic acid sequence has been Subcloned in an antisense nucleotides derived from the gene of interest, or from any orientation (i.e., RNA transcribed from the inserted nucleic nucleic acid capable of encoding an orthologue, paralogue or acid will be of an antisense orientation to a target nucleic acid homologue of the protein of interest) in a sense orientation of interest). Preferably, production of antisense nucleic acid into a plant. “Sense orientation” refers to a DNA sequence sequences in plants occurs by means of a stably integrated that is homologous to an mRNA transcript thereof. Intro nucleic acid construct comprising a promoter, an operably duced into a plant would therefore beat least one copy of the linked antisense oligonucleotide, and a terminator. nucleic acid sequence. The additional nucleic acid sequence 0090 The nucleic acid molecules used for silencing in the will reduce expression of the endogenous gene, giving rise to methods of the invention (whether introduced into a plant or a phenomenon known as co-suppression. The reduction of generated in situ) hybridize with or bind to mRNA transcripts gene expression will be more pronounced if several addi and/or genomic DNA encoding a polypeptide to thereby tional copies of a nucleic acid sequence are introduced into inhibit expression of the protein, e.g., by inhibiting transcrip the plant, as there is a positive correlation between high tion and/or translation. The hybridization can be by conven transcript levels and the triggering of co-suppression. tional nucleotide complementarity to form a stable duplex, or, 0087 Another example of an RNA silencing method for example, in the case of an antisense nucleic acid sequence involves the use of antisense nucleic acid sequences. An which binds to DNA duplexes, through specific interactions 'antisense' nucleic acid sequence comprises a nucleotide in the major groove of the double helix. Antisense nucleic sequence that is complementary to a “sense' nucleic acid acid sequences may be introduced into a plant by transforma sequence encoding a protein, i.e. complementary to the cod tion or direct injection at a specific tissue site. ing strand of a double-stranded cDNA molecule or comple mentary to an mRNA transcript sequence. The antisense 0091 Alternatively, antisense nucleic acid sequences can nucleic acid sequence is preferably complementary to the be modified to target selected cells and then administered endogenous gene to be silenced. The complementarity may systemically. For example, for systemic administration, anti be located in the “coding region' and/or in the “non-coding sense nucleic acid sequences can be modified such that they region' of a gene. The term "coding region” refers to a region specifically bind to receptors or antigens expressed on a of the nucleotide sequence comprising codons that are trans selected cell Surface, e.g., by linking the antisense nucleic lated into amino acid residues. The term “non-coding region” acid sequence to peptides or antibodies which bind to cell refers to 5' and 3' sequences that flank the coding region that surface receptors or antigens. The antisense nucleic acid are transcribed but not translated into amino acids (also sequences can also be delivered to cells using the vectors referred to as 5' and 3' untranslated regions). described herein. 0088 Antisense nucleic acid sequences can be designed 0092. According to a further aspect, the antisense nucleic according to the rules of Watson and Crick base pairing. The acid sequence is an a-anomeric nucleic acid sequence. An antisense nucleic acid sequence may be complementary to the a-anomeric nucleic add sequence forms specific double entire nucleic acid sequence (in this case a stretch of Substan stranded hybrids with complementary RNA in which, con tially contiguous nucleotides derived from the gene of inter trary to the usual b-units, the strands run parallel to each other est, or from any nucleic acid capable of encoding an ortho (Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The logue, paralogue or homologue of the protein of interest), but antisense nucleic acid sequence may also comprise a 2'-O- may also be an oligonucleotide that is antisense to only a part methylribonucleotide (Inoue et al. (1987) Nucl Ac Res 15, of the nucleic acid sequence (including the mRNA 5' and 3' 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. UTR). For example, the antisense oligonucleotide sequence (1987) FEBS Lett. 215,327-330). may be complementary to the region Surrounding the trans 0093. The reduction or substantial elimination of endog lation start site of an mRNA transcript encoding a polypep enous gene expression may also be performed using tide. The length of a Suitable antisense oligonucleotide ribozymes. Ribozymes are catalytic RNA molecules with sequence is known in the art and may start from about 50, 45. ribonuclease activity that are capable of cleaving a single 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An Stranded nucleic acid sequence. Such as an mRNA, to which antisense nucleic acid sequence according to the invention they have a complementary region. Thus, ribozymes (e.g., may be constructed using chemical synthesis and enzymatic hammerhead ribozymes (described in Haselhoff and Gerlach ligation reactions using methods known in the art. For (1988) Nature 334, 585-591) can be used to catalytically example, an antisense nucleic acid sequence (e.g., an anti cleave mRNA transcripts encoding a polypeptide, thereby sense oligonucleotide sequence) may be chemically synthe substantially reducing the number of mRNA transcripts to be sized using naturally occurring nucleotides or variously translated into a polypeptide. A ribozyme having specificity modified nucleotides designed to increase the biological sta for a nucleic acid sequence can be designed (see for example: bility of the molecules or to increase the physical stability of Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. the duplex formed between the antisense and sense nucleic No. 5,116,742). Alternatively, mRNA transcripts correspond acid sequences, e.g., phosphorothioate derivatives and acri ing to a nucleic acid sequence can be used to select a catalytic dine substituted nucleotides may be used. Examples of modi RNA having a specific ribonuclease activity from a pool of fied nucleotides that may be used to generate the antisense RNA molecules (Bartel and Szostak (1993) Science 261, nucleic acid sequences are well known in the art. Known 141 1-1418). The use of ribozymes for gene silencing in plants nucleotide modifications include methylation, cyclization is known in the art (e.g., Atkins et al. (1994) WO94/00012: and caps and substitution of one or more of the naturally Lenne et al. (1995) WO95/03404; Lutziger et al. (2000) WO occurring nucleotides with an analogue Suchasinosine. Other 00/00619; Prinsen et al. (1997) WO97/13865 and Scott et al. modifications of nucleotides are well known in the art. (1997) WO 97/38116). US 2015/0106974 A1 Apr. 16, 2015

0094 Gene silencing may also be achieved by insertion for design and generation of amiRNAS and their precursors mutagenesis (for example, T-DNA insertion or transposon are also available to the public (Schwab et al., Plant Cell 18, insertion) or by strategies as described by, among others, 1121-1133, 2006). Angell and Baulcombe (1999) Plant J 20(3): 357-62), (Am 0101 For optimal performance, the gene silencing tech plicon VIGS WO 98/36083), or Baulcombe (WO 99/15682). niques used for reducing expression in a plant of an endog 0095 Gene silencing may also occur if there is a mutation enous gene requires the use of nucleic acid sequences from on an endogenous gene and/or a mutation on an isolated monocotyledonous plants for transformation of monocotyle gene/nucleic acid Subsequently introduced into a plant. The donous plants, and from dicotyledonous plants for transfor reduction or Substantial elimination may be caused by a non mation of dicotyledonous plants. Preferably, a nucleic acid functional polypeptide. For example, the polypeptide may sequence from any given plant species is introduced into that bind to various interacting proteins; one or more mutation(s) same species. For example, a nucleic acid sequence from rice and/or truncation(s) may therefore provide for a polypeptide is transformed into a rice plant. However, it is not an absolute that is still able to bind Interacting proteins (such as receptor requirement that the nucleic acid sequence to be introduced proteins) but that cannot exhibit its normal function (such as originates from the same plant species as the plant in which it signalling ligand). will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the 0096. A further approach to gene silencing is by targeting nucleic acid to be introduced. nucleic acid sequences complementary to the regulatory 0102 Described above are examples of various methods region of the gene (e.g., the promoter and/or enhancers) to for the reduction or Substantial elimination of expression in a form triple helical structures that prevent transcription of the plant of an endogenous gene. A person skilled in the art would gene in target cells. See Helene, C., Anticancer Drug Res. 6. readily be able to adapt the aforementioned methods for 569-84, 1991; Helene et al., Ann. N.Y. Acad. Sc. 660, 27-36 silencing so as to achieve reduction of expression of an 1992; and Maher, L. J. Bioassays 14,807-15, 1992. endogenous gene in a whole plant or in parts thereof through 0097. Other methods, such as the use of antibodies the use of an appropriate promoter, for example. directed to an endogenous polypeptide for inhibiting its func tion in planta, or interference in the signalling pathway in Transformation which a polypeptide is involved, will be well known to the (0103. The term “introduction' or “transformation as skilled man. In particular, it can be envisaged that manmade referred to herein encompasses the transfer of an exogenous molecules may be useful for inhibiting the biological function polynucleotide into a host cell, irrespective of the method of a target polypeptide, or for interfering with the signalling used for transfer. Plant tissue capable of subsequent clonal pathway in which the target polypeptide is involved. propagation, whether by organogenesis or embryogenesis, 0098. Alternatively, a screening program may be set up to may be transformed with a genetic construct of the present identify in a plant population natural variants of a gene, which invention and a whole plant regenerated there from. The variants encode polypeptides with reduced activity. Such particular tissue chosen will vary depending on the clonal natural variants may also be used for example, to perform propagation systems available for, and best Suited to, the homologous recombination. particular species being transformed. Exemplary tissue tar gets include leaf disks, pollen, embryos, cotyledons, hypoco 0099 Artificial and/or natural microRNAs (miRNAs) tyls, megagametophytes, callus tissue, existing meristematic may be used to knock out gene expression and/or mRNA tissue (e.g., apical meristem, axillary buds, and root mer translation. Endogenous miRNAS are single stranded Small istems), and induced meristem tissue (e.g., cotyledon mer RNAs of typically 19-24 nucleotides long. They function istem and hypocotyl meristem). The polynucleotide may be primarily to regulate gene expression and/l or mRNA trans transiently or stably introduced into a host cell and may be lation. Most plant microRNAs (miRNAs) have perfect or maintained non-integrated, for example, as a plasmid. Alter near-perfect complementarity with their target sequences. natively, it may be integrated into the host genome. The However, there are natural targets with up to five mismatches. resulting transformed plant cell may then be used to regener They are processed from longer non-coding RNAS with char ate a transformed plant in a manner known to persons skilled acteristic fold-back structures by double-strand specific in the art. RNases of the Dicer family. Upon processing, they are incor 0104. The transfer of foreign genes into the genome of a porated in the RNA-induced silencing complex (RISC) by plant is called transformation. Transformation of plant spe binding to its main component, an Argonaute protein. MiR cies is now a fairly routine technique. Advantageously, any of NAS serve as the specificity components of RISC, since they several transformation methods may be used to introduce the base-pair to target nucleic acids, mostly mRNAS, in the cyto gene of interest into a suitable ancestor cell. The methods plasm. Subsequent regulatory events include target mRNA described for the transformation and regeneration of plants cleavage and destruction and/or translational inhibition. from plant tissues or plant cells may be utilized for transient Effects of miRNA overexpression are thus often reflected in or for stable transformation. Transformation methods include decreased mRNA levels of target genes. the use of liposomes, electroporation, chemicals that increase 0100 Artificial microRNAs (amiRNAs), which are typi free DNA uptake, injection of the DNA directly into the plant, cally 21 nucleotides in length, can be genetically engineered particle gun bombardment, transformation using viruses or specifically to negatively regulate gene expression of single pollen and microprojection. Methods may be selected from or multiple genes of interest. Determinants of plant the calcium/polyethylene glycol method for protoplasts microRNA target selection are well known in the art. Empiri (Krens, F. A. et al., (1982) Nature 296,72-74; Negrutiu Iatal. cal parameters for target recognition have been defined and (1987) Plant Mol Biol 8:363-373); electroporation of proto can be used to aid in the design of specific amiRNAs, plasts (Shillito R. D. at al. (1985) Bio/Technol 3. 1099-1102); (Schwab et al., Dev. Cell 8,517-527, 2005). Convenient tools microinjection into plant material (Crossway A et al., (1986) US 2015/0106974 A1 Apr. 16, 2015

Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle obtained at a later point in time (Chang (1994). Plant J. 5: bombardment (Klein T M at al., (1987) Nature 327: 70) 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). infection with (non-integrative) viruses and the like. Trans However, an especially effective method is the vacuum infil genic plants, including transgenic crop plants, are preferably tration method with its modifications such as the “floral dip' produced via Agrobacterium-mediated transformation. An method. In the case of vacuum infiltration of Arabidopsis, advantageous transformation method is the transformation in intact plants under reduced pressure are treated with an agro planta. To this end, it is possible, for example, to allow the bacterial suspension Bechthold, N (1993). C R Acad Sci agrobacteria to act on plant seeds or to inoculate the plant Paris Life Sci, 316; 1194-1199, while in the case of the meristem with agrobacteria. It has proved particularly expe “floral dip' method the developing floral tissue is incubated dient in accordance with the invention to allow a suspension briefly with a Surfactant-treated agrobacterial Suspension of transformed agrobacteria to act on the intact plant or at Cough, SJ and Bent A F (1998) The Plant J. 16,735-743). A least on the flower primordia. The plant is subsequently certain proportion of transgenic seeds are harvested in both grown on until the seeds of the treated plant are obtained cases, and these seeds can be distinguished from non-trans (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for genic seeds by growing under the above-described selective Agrobacterium-mediated transformation of rice include well conditions. In addition the stable transformation of plastids is known methods for rice transformation, such as those of advantages because plastids are inherited maternally is described in any of the following: European patent applica most crops reducing or eliminating the risk of transgene flow tion EP 1198985 A1, Aldemita and Hodges (Planta 199: through pollen. The transformation of the chloroplast genome 612-617, 1996); Chaneta (Plant Mol Biol 22 (3): 491-506, is generally achieved by a process which has been schemati 1993), Hiel at al. (Plant J 6 (2): 271-282, 1994), which dis cally displayed in Klaus et al., 2004 Nature Biotechnology closures are incorporated by reference herein as if fully set 22 (2), 225-229. Briefly the sequences to be transformed are forth. In the case of corn transformation, the preferred method cloned together with a selectable marker gene between flank is as described in either Ishida et al. (Nat. Biotechnol 14(6): ing sequences homologous to the chloroplast genome. These 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, homologous flanking sequences direct site specific integra 2002), which disclosures are incorporated by reference tion into the plastome. Plastidal transformation has been herein as if fully set forth. Said methods are further described described for many different plant species and an overview is by way of example in B. Jenes et al., Techniques for Gene given in Bock (2001) Transgenic plastids in basic research Transfer, in: Transgenic Plants, Vol. 1, Engineering and Uti and plant biotechnology. J Mol Biol. 2001 Sep. 21: 312 (3): lization, eds. S. D. Kung and R. Wu, Academic Press (1993) 425-38 or Maliga, P (2003) Progress towards commercializa 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant tion of plastid transformation technology. Trends Biotechnol. Molec. Biol. 42 (1991) 205-225). The nucleic acids or the 21, 20-28. Further biotechnological progress has recently construct to be expressed is preferably cloned into a vector, been reported in form of marker free plastid transformants, which is suitable for transforming Agrobacterium tumefa which can be produced by a transient co-integrated maker ciens, for example plBin 19 (Bevan et al., Nucl. Acids Res. 12 gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225 (1984) 8711). Agrobacteria transformed by such a vector can 229). then be used in known manner for the transformation of 0106 The genetically modified plant cells can be regen plants, such as plants used as a model, like Arabidopsis (Ara erated via all methods with which the skilled worker is famil bidopsis thaliana is within the scope of the present invention iar. Suitable methods can be found in the abovementioned not considered as a crop plant), or crop plants such as, by way publications by S. D. Kung and R. Wu, Potrykus or Höfgen of example, tobacco plants, for example by immersing and Willimitzer. bruised leaves or chopped leaves in an agrobacterial Solution 0107 Generally after transformation, plant cells or cell and then culturing them in Suitable media. The transformation groupings are selected for the presence of one or more mark of plants by means of Agrobacterium tumefaciens is ers which are encoded by plant-expressible genes co-trans described, for example, by Höfgen and Willimitzer in Nucl. ferred with the gene of interest, following which the trans Acid Res. (1988) 16, 9877 or is known interalia from F. F. formed material is regenerated into a whole plant. To select White, Vectors for Gene Transfer in Higher Plants; in Trans transformed plants, the plant material obtained in the trans genic Plants, Vol. 1, Engineering and Utilization, eds. S. D. formation is, as a rule, Subjected to selective conditions so Kung and R. Wu, Academic Press, 1993, pp. 15-38. that transformed plants can be distinguished from untrans 0105. In addition to the transformation of somatic cells, formed plants. For example, the seeds obtained in the above which then have to be regenerated into intact plants, it is also described manner can be planted and, after an initial growing possible to transform the cells of plant meristems and in period, Subjected to a suitable selection by spraying. A further particular those cells which develop into gametes. In this possibility consists in growing the seeds, if appropriate after case, the transformed gametes follow the natural plant devel sterilization, on agar plates using a Suitable selection agent so opment, giving rise to transgenic plants. Thus, for example, that only the transformed seeds can grow into plants. Alter seeds of Arabidopsis are treated with agrobacteria and seeds natively, the transformed plants are screened for the presence are obtained from the developing plants of which a certain of a selectable marker such as the ones described above. proportion is transformed and thus transgenic Feldman, KA 0108. Following DNA transfer and regeneration, puta and Marks M D (1987). Mol Gen Genet 208:274-289; Feld tively transformed plants may also be evaluated, for instance mann K (1992). In: C Koncz, N-H Chua and J Shell, eds. using Southern analysis, for the presence of the gene of inter Methods in Arabidopsis Research. Word Scientific, Sin est, copy number and/or genomic organisation. Alternatively gapore, pp. 274-289). Alternative methods are based on the or additionally, expression levels of the newly introduced repeated removal of the inflorescences and incubation of the DNA may be monitored using Northern and/or Western excision site in the center of the rosette with transformed analysis, both techniques being well known to persons having agrobacteria, whereby transformed seeds can likewise be ordinary skill in the art. US 2015/0106974 A1 Apr. 16, 2015

0109 The generated transformed plants may be propa Homologous Recombination gated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation 0112 Homologous recombination allows introduction in (or T1) transformed plant may be selfed and homozygous a genome of a selected nucleic acid at a defined selected second-generation (or T2) transformants selected, and the T2 position. Homologous recombination is a standard technol plants may then further be propagated through classical ogy used routinely in biological sciences for lower organisms breeding techniques. The generated transformed organisms such as yeast or the moss Physcomitrella. Methods for per may take a variety of forms. For example, they may be chi forming homologous recombination in plants have been meras of transformed cells and non-transformed cells; clonal described not only for model plants (Offringa et al. (1990) transformants (e.g., all cells transformed to contain the EMBOJ9(10): 3077-84) but also for crop plants, for example expression cassette); grafts of transformed and untrans rice (Terada et al. (2002)Nat Biotech 20(10): 1030-4:lida and Terada (2004) Curr Opin Biotech 15(2): 132-8), and formed tissues (e.g., in plants, a transformed rootstock approaches exist that are generally applicable regardless of grafted to an untransformed Scion). the target organism (Miller et al. Nature Biotechnol. 25, 778 785, 2007). T-DNA Activation Tagging Yield Related Traits 0110 T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually 0113. Yield related traits comprise one or more of yield, containing a promoter (may also be a translation enhancer or biomass, seed yield, early vigour, greenness index, increased an intron), in the genomic region of the gene of interest or 10 growth rate, improved agronomic traits (such as Improved kb up- or downstream of the coding region of a gene in a Water Use Efficiency (WUE), Nitrogen Use Efficiency configuration Such that the promoter directs expression of the (NUE), etc.). targeted gene. Typically, regulation of expression of the tar geted gene by its natural promoter is disrupted and the gene Yield falls under the control of the newly introduced promoter. The 0114. The term "yield' in general means a measurable promoter is typically embedded in a T-DNA. This T-DNA is produce of economic value, typically related to a specified randomly inserted into the plant genome, for example, crop, to an area, and to a period of time. Individual plant parts through Agrobacterium infection and leads to modified directly contribute to yield based on their number, size and/or expression of genes near the inserted T-DNA. The resulting weight, or the actual yield is the yield per square meter for a transgenic plants show dominant phenotypes due to modified crop and year, which is determined by dividing total produc expression of genes close to the introduced promoter. tion (includes both harvested and appraised production) by planted square meters. The term "yield of a plant may relate Tilling to vegetative biomass (root and/or shoot biomass), to repro ductive organs, and/or to propagules (such as seeds) of that 0111. The term “TILLING” is an abbreviation of “Tar plant. geted Induced Local Lesions in Genomes and refers to a 0115 Taking corn as an example, a yield increase may be mutagenesis technology useful to generate and/or identify manifested as one or more of the following: increase in the nucleic acids encoding proteins with modified expression number of plants established per square meter, an increase in and/or activity. TILLING also allows selection of plants car the number of ears per plant, an increase in the number of rying Such mutant variants. These mutant variants may rows, number of kernels per row, kernel weight, thousand exhibit modified expression, either in strength or in location kernel weight, earlength/diameter, increase in the seed filling or in timing (if the mutations affect the promoter for rate (which is the number of filled seeds divided by the total example). These mutant variants may exhibit higher activity number of seeds and multiplied by 100), among others. Tak than that exhibited by the gene in its natural form. TILLING ing rice as an example, a yield increase may manifest itself as combines high-density mutagenesis with high-throughput an increase in one or more of the following: number of plants screening methods. The steps typically followed in TILLING per square meter, number of panicles per plant, panicle are: (a) EMS mutagenesis (Redei GP and Koncz C (1992) In length, number of spikelets per panicle, number of flowers Methods in Arabidopsis Research, Koncz. C. Chua N H. (florets) per panicle, increase in the seed filling rate (which is Schell J, eds. Singapore, World Scientific Publishing Co., pp. the number of filled seeds divided by the total number of 16-82; Feldmann et al., (1994) In Meyerowitz E. M. Somer seeds and multiplied by 100), increase in thousand kernel ville CR, eds, Arabidopsis. Cold Spring Harbor Laboratory weight, among others. In rice, Submergence tolerance may Press, Cold Spring Harbor, N.Y., pp 137-172: Lightner J and also result in increased yield. Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Meth ods on Molecular Biology, Vol. 82. Humana Press, Totowa, Early Vigour N.J., pp. 91-104); (b) DNA preparation and pooling of indi viduals; (c) PCR amplification of a region of interest; (d) 0116 “Early vigour' refers to active healthy well-bal denaturation and annealing to allow formation of heterodu anced growth especially during early stages of plant growth, plexes; (e) DHPLC, where the presence of a heteroduplex in and may result from increased plant fitness due to, for a pool is detected as an extra peak in the chromatogram, (f) example, the plants being better adapted to their environment identification of the mutant individual; and (g) sequencing of (i.e. optimizing the use of energy resources and partitioning the mutant PCR product. Methods for TILLING are well between shoot and root). Plants having early vigour also show known in the art (McCallum et al., (2000) Nat Biotechnol 18: increased seedling survival and a better establishment of the 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): crop, which often results in highly uniform fields (with the 145-50). crop growing in uniform manner, i.e. with the majority of US 2015/0106974 A1 Apr. 16, 2015

plants reaching the various stages of development at Substan stressed plants of less than 40%, 35%, 30% or 25%, more tially the same time), and often better and higher yield. There preferably less than 20% or 15% in comparison to the control fore, early vigour may be determined by measuring various plant under non-stress conditions. Due to advances in agri factors, such as thousand kernel weight, percentage germina cultural practices (irrigation, fertilization, pesticide treat tion, percentage emergence, seedling growth, seedling ments) severe stresses are not often encountered in cultivated height, root length, root and shoot biomass and many more. crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for Increased Growth Rate agriculture. Mild stresses are the everyday biotic and/or abi otic (environmental) stresses to which a plant is exposed. 0117 The increased growth rate may be specific to one or Abiotic stresses may be due to drought or excess water, more parts of a plant (including seeds), or may be throughout anaerobic stress, salt stress, chemical toxicity, oxidative Substantially the whole plant. Plants having an increased stress and hot, cold or freezing temperatures. The abiotic growth rate may have a shorter life cycle. The life cycle of a stress may be an osmotic stress caused by a water stress plant may be taken to mean the time needed to grow from a (particularly due to drought), salt stress, oxidative stress oran dry mature seed up to the stage where the plant has produced ionic stress. Biotic stresses are typically those stresses caused dry mature seeds, similar to the starting material. This life by pathogens, such as bacteria, viruses, fungi, nematodes and cycle may be influenced by factors such as speed of germi insects. nation, early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth 0119. In particular, the methods of the present invention rate may take place at one or more stages in the life cycle of a may be performed under non-stress conditions or under con plant or during substantially the whole plant life cycle. ditions of mild drought to give plants having increased yield Increased growth rate during the early stages in the life cycle relative to control plants. As reported in Wang et al. (Planta of a plant may reflectenhanced vigour. The increase in growth (2003) 218: 1-14), abiotic stress leads to a series of morpho rate may alter the harvest cycle of a plantallowing plants to be logical, physiological, biochemical and molecular changes sown later and/or harvested sooner than would otherwise be that adversely affect plant growth and productivity. Drought, possible (a similar effect may be obtained with earlier flow salinity, extreme temperatures and oxidative stress are known ering time). If the growth rate is sufficiently increased, it may to be interconnected and may induce growth and cellular allow for the further sowing of seeds of the same plant species damage through similar mechanisms. Rabbani et al. (Plant (for example sowing and harvesting of rice plants followed by Physiol (2003) 133: 1755-1767) describes a particularly high sowing and harvesting of further rice plants all within one degree of “cross talk” between drought stress and high-salin conventional growing period). Similarly, if the growth rate is ity stress. For example, drought and/or salinisation are mani sufficiently increased, it may allow for the further sowing of fested primarily as osmotic stress, resulting in the disruption seeds of different plants species (for example the sowing and of homeostasis and ion distribution in the cell. Oxidative harvesting of corn plants followed by, for example, the Sow stress, which frequently accompanies high or low tempera ing and optional harvesting of soybean, potato or any other ture, Salinity or drought stress, may cause denaturing of func Suitable plant). Harvesting additional times from the same tional and structural proteins. As a consequence, these diverse rootstock in the case of some crop plants may also be pos environmental stresses often activate similar cell signalling sible. Altering the harvest cycle of a plant may lead to an pathways and cellular responses, such as the production of increase in annual biomass production per square meter (due stress proteins, up-regulation of anti-oxidants, accumulation to an increase in the number of times (say in a year) that any of compatible solutes and growth arrest. The term “non particular plant may be grown and harvested). An increase in stress' conditions as used herein are those environmental growth rate may also allow for the cultivation of transgenic conditions that allow optimal growth of plants. Persons plants in a wider geographical area than their wild-type coun skilled in the art are aware of normal soil conditions and terparts, since the territorial limitations for growing a crop are climatic conditions for a given location. Plants with optimal often determined by adverse environmental conditions either growth conditions, (grown under non-stress conditions) typi at the time of planting (early season) or at the time of harvest cally yield in increasing order of preference at least 97%, ing (late season). Such adverse conditions may be avoided if 95%, 92%, 90%, 87%, 85%, 83%, 80%, 77% or 75% of the the harvest cycle is shortened. The growth rate may be deter average production of Such plant in a given environment. mined by deriving various parameters from growth curves, Average production may be calculated on harvest and/or sea such parameters may be: T-Mid (the time taken for plants to son basis. Persons skilled in the art are aware of average yield reach 50% of their maximal size) and T-90 (time taken for productions of a crop. plants to reach 90% of their maximal size), amongst others. I0120 Nutrient deficiency may result from a lack of nutri ents such as nitrogen, phosphates and other phosphorous Stress Resistance containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others. 0118. An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether 0.121. The term salt stress is not restricted to common salt the plant is exposed to various stresses compared to control (NaCl), but may be any one or more of NaCl, KC1, LiCl, plants. Plants typically respond to exposure to stress by grow MgCl2, CaCl2, amongst others. ing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is Increase/Improve/Enhance defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether 0.122 The terms “increase”, “improve' or "enhance' are without the capacity to resume growth. Mild stress in the interchangeable and shall mean in the sense of the application sense of the invention leads to a reduction in the growth of the at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at US 2015/0106974 A1 Apr. 16, 2015

least 15% or 20%, more preferably 25%, 30%, 35% or 40% length. These nucleic acids may be used as restriction frag more yield and/or growth in comparison to control plants as ment length polymorphism (RFLP) markers. Southern blots defined herein. (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant Seed Yield genomic DNA may be probed with the ISYT-LIKE-encoding 0123 Increased seed yield may manifest itself as one or nucleic acids. The resulting banding patterns may then be more of the following: a) an increase in seed biomass (total Subjected to genetic analyses using computer programs such seed weight) which may be on an individual seed basis and/or as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in per plant and/or per square meter; b) increased number of order to construct a genetic map. In addition, the nucleic acids flowers per plant; c) increased number of (filled) seeds; d) may be used to probe Southern blots containing restriction increased seed filling rate (which is expressed as the ratio endonuclease-treated genomic DNAs of a set of individuals between the number of filled seeds divided by the total num representing parent and progeny of a defined genetic cross. ber of seeds); e) increased harvest index, which is expressed Segregation of the DNA polymorphisms is noted and used to as a ratio of the yield of harvestable parts, such as seeds, calculate the position of the iSYT-like polypeptide-encoding divided by the total biomass; and f) increased thousand kernel nucleic acid in the genetic map previously obtained using this weight (TKW), which is extrapolated from the number of population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314 331). filled seeds counted and their total weight. An increased TKW I0128. The production and use of plant gene-derived may result from an increased seed size and/or seed weight, probes for use in genetic mapping is described in Bernatzky and may also result from an increase in embryo and/or and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. endosperm size. Numerous publications describe genetic mapping of specific 0.124. An increase in seed yield may also be manifested as cDNA clones using the methodology outlined above or varia an increase in seed size and/or seed Volume. Furthermore, an tions thereof. For example, F2 intercross populations, back increase in seed yield may also manifest itself as an increase cross populations, randomly mated populations, near in seed area and/or seed length and/or seed width and/or seed isogenic lines, and other sets of individuals may be used for perimeter. Increased yield may also result in modified archi mapping. Such methodologies are well known to those tecture, or may occur because of modified architecture. skilled in the art. Greenness Index I0129. The nucleic acid probes may also be used for physi cal mapping (i.e., placement of sequences on physical maps: 0.125. The “greenness index' as used herein is calculated see Hoheisel et al. In: Non-mammalian Genomic Analysis: A from digital images of plants. For each pixel belonging to the Practical Guide, Academic press 1996, pp. 319-346, and ref plant object on the image, the ratio of the green value Versus erences cited therein). the red value (in the RGB model for encoding color) is cal 0.130. In another embodiment, the nucleic acid probes may culated. The greenness index is expressed as the percentage of be used in direct fluorescence in situ hybridisation (FISH) pixels for which the green-to-red ratio exceeds a given thresh mapping (Trask (1991) Trends Genet. 7:149-154). Although old. Under normal growth conditions, under salt stress growth current methods of FISH mapping favour use of large clones conditions, and under reduced nutrient availability growth (several kb to several hundred kb; see Laan et al. (1995) conditions, the greenness index of plants is measured in the Genome Res. 5:13-20), improvements in sensitivity may last imaging before flowering. In contrast, under drought allow performance of FISH mapping using shorter probes. stress growth conditions, the greenness index of plants is I0131) A variety of nucleic acid amplification-based meth measured in the first imaging after drought. ods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific Marker Assisted Breeding amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), 0126 Such breeding programmes sometimes require polymorphism of PCR-amplified fragments (CAPS; Shef introduction of allelic variation by mutagenic treatment of the field etal. (1993) Genomics 16:325-332), allele-specific liga plants, using for example EMS mutagenesis; alternatively, tion (Landegren et al. (1988) Science 241:1077-1080), nucle the programme may start with a collection of allelic variants otide extension reactions (Sokolov (1990) Nucleic Acid Res. of so called “natural origin caused unintentionally. Identifi 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) cation of allelic variants then takes place, for example, by Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook PCR. This is followed by a step for selection of superior (1989) Nucleic Acid Res. 17:6795-6807). For these methods, allelic variants of the sequence in question and which give the sequence of a nucleic acid is used to design and produce increased yield. Selection is typically carried out by monitor primer pairs for use in the amplification reaction or in primer ing growth performance of plants containing different allelic extension reactions. The design of such primers is well known variants of the sequence in question. Growth performance to those skilled in the art. In methods employing PCR-based may be monitored in a greenhouse or in the field. Further genetic mapping, it may be necessary to identify DNA optional steps include crossing plants in which the Superior sequence differences between the parents of the mapping allelic variant was identified with another plant. This could be cross in the region corresponding to the instant nucleic acid used, for example, to make a combination of interesting phe sequence. This, however, is generally not necessary for map notypic features. ping methods. Use as Probes in (Gene Mapping) Plant 0127. Use of nucleic acids encoding the protein of interest 0.132. The term “plant’ as used herein encompasses whole for genetically and physically mapping the genes requires plants, ancestors and progeny of the plants and plant parts, only a nucleic acid sequence of at least 15 nucleotides in including seeds, shoots, stems, leaves, roots (including US 2015/0106974 A1 Apr. 16, 2015

tubers), flowers, and tissues and organs, wherein each of the Triticum hybernum, Triticum macha, Triticum sativum, Triti aforementioned comprise the gene/nucleic acid of interest. cum monococcum or Triticum vulgare), Tropaeolum minus, The term "plant” also encompasses plant cells, Suspension Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., cultures, callus tissue, embryos, meristematic regions, game Viola Odorata, Vitis spp., Zee mays, Zizania palustris, Zizi tophytes, sporophytes, pollen and microspores, again phus spp., amongst others. wherein each of the aforementioned comprises the gene/ Control Plant(s) The choice of suitable control plants is a nucleic acid of interest. routine part of an experimental setup and may include corre sponding wildtype plants or corresponding plants without the 0.133 Plants that are particularly useful in the methods of gene of interest. The control plant is typically of the same the invention include all plants which belong to the superfam plant species or even of the same variety as the plant to be ily Viridiplantae, in particular monocotyledonous and dicoty assessed. The control plant may also be a nullizygote of the ledonous plants including fodder or forage legumes, orna plant to be assessed. Nullizygotes are individuals missing the mental plants, food crops, trees or shrubs selected from the transgene by segregation. A "control plant as used herein list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium refers not only to whole plants, but also to plant parts, includ spp., Amaranthus spp., Ammophila arenaria, Ananas Como ing seeds and seed parts. sus, Annona spp., Apium graveolens, Arachis spp., Artocarpus DETAILED DESCRIPTION OF THE INVENTION spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena I0134) Surprisingly, it has now been found that modulating hybrida), Averrhoa carambola, Bambusa sp., Benincasa his expression in a plant of one or more nucleic acid(s) encoding pida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. at least two iSYT polypeptides selected from the group con Brassica napus, Brassica rapa Ssp. canola, oilseed rape, sisting of any of the polypeptides of Table A, homologues turnip rape), Cadaba farinosa, Camelia sinensis, Canna thereof and fusions of the same gives plants having enhanced indica, Cannabis sativa, Capsicum spp., Carexelata, Carica yield-related traits relative to control plants. According to a papaya, Carissa macrocarpa, Carya spp., Carthamus tincto first embodiment, the present invention provides a method for rius, Castanea spp., Celba pentandra, Cichorlum endvia, enhancing yield-related traits in plants relative to control Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos plants, comprising modulating expression in a plant of one or spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus more nucleic acid(s) encoding at least two iSYT polypeptides sp., Coriandrum sativum, Corylus spp., Crataegus spp., Cro selected from the group consisting of any of the polypeptides cus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Dau of Table A, homologues thereof and fusions of the same and cus carota, Desmodium spp., Dimocarpus longan, Dioscorea optionally selecting for plants having enhanced yield-related spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis traits. guineensis, Elaeis Oleifera), Eleusine coracana, Eragrostis 0.135 A preferred method for modulating, increasing or tef, Erianthus sp., Erioborya japonica, Eucalyptus sp., Euge decreasing expression of a nucleic acid encoding at least two nia uniflora, Fagopyrum spp., Fagus spp., Festuca arundina iSYT polypeptides selected from the group consisting of any cea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo of the polypeptides of Table A, homologues thereof and biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja fusions of the same is by introducing and expressing in a plant max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus a nucleic acid encoding a iSYT-like polypeptide. annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. 0.136 Any reference hereinafter to a “protein useful in the (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lac methods of the invention' is taken to mean an iSYT polypep tuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissi tide, homologue thereof or fusions of the same as defined mum, Litchi chinensis, Lotus spp., Lufa acutangula, Lupinus herein. Any reference hereinafter to a “nucleic acid useful in spp., Luzula Sylvatica, Lycopersicon spp. (e.g. Lycopersicon the methods of the invention' is taken to mean a nucleic acid esculentum, Lycopersicon lycopersicum, Lycopersicon pyri capable of encoding Such an iSYT polypeptide, homologue forme), Macrotyloma spp., Malus spp., Malpighia emargi– thereof or fusions of the same. The nucleic acid to be intro nata, Mammea americana, Mangifera indica, Manihot spp., duced into a plant (and therefore useful in performing the Manilkara Zapota, Medicago sativa, Melilotus spp., Mentha methods of the invention) is any nucleic acid encoding the spp., Miscanthus sinensis, Momordica spp., Morus nigra, type of protein which will now be described, hereafter also Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornitho named "iSYT nucleic acid” or “iSYT gene'. pus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), I0137. An "iSYT polypeptide' as defined herein refers to Panicum miliaceum, Panicum virgatum, Passiflora edulis, any of the polypeptide of Table A and maybe represented by Pastninaca sativa, Pennisetum sp., Persea spp., Peroselinum the corresponding amino acid sequence as provided in the crispum, Phalaris arundinacea, Phaseolus spp., Phleum pre sequence listing. Further description of an iSYT polypeptide tense, Phoenix spp., Phragmites australis, Physalis spp., is provided in Table B. Pinus spp., Pistacia vera, Pisum spp., Poe spp., Populus spp., I0138 An "iSYT-like polypeptide' as defined herein refers Prosopis spp., Prunus spp., Psidium spp., Punica granatum, to any polypeptide selected from the group consisting of any Pyrus communis, Quercus spp., Raphanus sativus, Rheum of the polypeptides of Table A, homologues thereof and rharbararum, Ribes spp., Ricinus communis, Rubus spp., fusions of the same. Polypeptides useful in the methods of the Saccharum spp., Salix sp., Sambucus spp., Secale cereale, invention are iSYT polypeptides as well as iSYT-like Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum polypeptides. tuberosum, Solanum integrifolium or Solanum lycopersi (0.139. A fusion of iSYT and/or iSYT-like polypeptides is cum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes preferably encoded by a nucleic acid which may be con spp., Tamarindus indica, Theobroma cacao, Triflium spp., structed using well known recombinant DNA techniques Tripsacum dactyloides, Triticosecale rinpaui, Triticum spp. (Sambrook J, Fritsch E F and Manlatis T (1989) Molecular (e.g. Triticum aestivum, Triticum durum, Tritium turgidum, Cloning, A Laboratory Manual). For example the protein US 2015/0106974 A1 Apr. 16, 2015

fusion may comprise a fusion of the entire iSYT and/or iSYT like polypeptides or of only a portion of the same, for example SEO ID NO: 67O : the N-terminal or the C-terminal portion. Preferably the por IOQYLDENKSLILKIVESQNSGKLSECAENOARLQRNLMYLAAIAD tion comprises one or more of the conserved domain corre 0145 Preferred homologues of a SYT polypeptide useful sponding to the domains of Tables C1 to C20 of the corre in the methods of the invention are listed in Table A2. sponding iSYT polypeptide. 0146 Preferably the methods of the invention concern a homologue of a SYT polypeptide derived from a crop plant more preferably, in increasing order of preference from corn, Concerning SYT Sugar cane, soybean, wheat, cotton and canola. 0147 Additionally or alternatively, the homologue of a 0140 SYT as defined herein refers to the polypeptide SYT polypeptide useful in the method in the invention has in encoded by the AN3 (ANGUSTIFOLIA 3) gene of Arabi increasing order of preference at least 25%, 26%. 27%, 28%, dopsis thaliana. Alternative names for the AN3 gene are GIF1 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, gene and SYT1 gene. The genomic locus and the AGI refer 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, ence of the SYT gene is AT59g28640. 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%. 65%, 66%, 67%, 68%, 0141. The terms “SYT, “SYT1 and “AN3” as used 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, herein are interchangeable. 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 0142. A “SYT polypeptide' as referred herein is repre 99% or 100% overall sequence identity to the amino acid sented by the sequence: represented by any one of the polypeptide sequences of Table A2 or of the SYT Polypeptide. The overall sequence identity (SEO ID NO: 671) is determined using a global alignment algorithm, Such as the MOOHLMOMOPMMAGYYPSNWTSDHIOQYLDENSKLILKIVESQNSGKLSE Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package. Accelrys), preferably with default CAENOARLQRNLMYLAAIADSQPQPPSVHSQYGSAGGGMIOGEGGSHYLQ parameters and preferably with sequences of mature proteins (i.e. without taking into account Secretion signals or transit QQQATOOOOMTOQSLMAARSSMLYAQQQQQQOPYATLOHOOLHHSQLGMS peptides). Compared to overall sequence identity, the SSSGGGGSSGLHILOGEAGGFHDFGRGKPEMGSGGGGEGRGGSSGDGGET sequence identity will generally be higher when only con served domains or motifs are considered. LYLKSSDDGN 0148 Preferably an iSYT, a homologue thereofor a fusion of the same useful in the methods of the invention comprises 0143 A “SYT Polypeptide' useful in the methods of the a domain having in increasing order of preference at least invention is preferably encoded by the following nucleic acid: 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, (SEO ID NO: 672) ATGCAACAGCACCTGATGCAGATGCAGCCCATGATGGCTGGTTACTACC 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or CCAGCAATGTTACCTCTGATCATATCCAACAGTACTTGGACGAAAACAA 100% sequence identity to the amino acid sequence of one or more of the conserved domain located at specific amino acid ATCGTTGATTCTGAAGATTGTTGAGTCTCAAAACTCTGGAAAGCTTAGC coordinates of an iSYT polypeptide according to Tables C1 to GAATGCGCCGAGAATCAAGCAAGGCTTCAACGCAACCTAATGTACCTAG C20, preferably to the domain identified in the HMMSmart database. CTGCAATAGCAGATTCTCAGCCTCAGCCACCAAGTGTGCATAGCCAGTA 0149 For Example, a homologue of the iSYTAT1G05370 polypeptide useful in the methods of the invention comprises TGGATCTGCTGGTGGTGGGATGATTCAGGGAGAAGGAGGGTCACACTAT a domain having in increasing order of preference at least TTGCAGCAGCAACAAGCGACT CAACAGCAACAGATGACTCAGCAGTCTC 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, TAATGGCGGCTCGATCTTCAATGTTGTATGCTCAGCAACAGCAGCAGCA 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, GCAGCCTTACGCGACGCTTCAGCATCAGCAATTGCACCATAGCCAGCTT 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or GGAATGAGCTCGAGCAGCGGAGGAGGAGGAAGCAGTGGTCTCCATATCC 100% sequence identity to the amino acid sequence of any TTCAGGGAGAGGCTGGTGGGTTTCATGATTTTGGCCGTGGGAAGCCGGA one or more of the conserved domains according to Table C1, preferably to the domain SMO0516 located at amino acid AATGGGAAGTGGTGGTGGCGGTGAAGGCAGAGGAGGAAGTTCAGGGGAT coordinates 86-229 of the iSYTAT1G05370 polypeptide. 0150 Preferred homologues of an iSYT polypeptide use GGTGGAGAAACCCTTTACTTGAAATCATCAGATGATGGGAATTGA ful in the methods of the invention are given in Tables A1 to A26. 0144. Alternatively or additionally, the term “SYT 0151. Additionally or alternatively, the homologue of a polypeptide or homologue thereofas defined herein refers to iSYT polypeptide useful in the method in the invention has in a polypeptide comprising an SNH domain having in increas increasing order of preference at least 25%, 26%. 27%, 28%, ing order of preference at least 40%, 45%, 50%, 55%, 60%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, domain of SEQID NO: 670. 59%, 60%, 61%, 62%, 63%, 64%. 65%, 66%, 67%, 68%, US 2015/0106974 A1 Apr. 16, 2015 20

69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, AT2G28290, AT1G21700, AT5G14170, AT4G17330, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, AT4G27550, AT1G65980, AT5G55210. AT3G15000, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, AT4G35550, AT1G20670, AT1G08730, AT5G13030, 99% or 100% overall sequence identity to the amino acid AT2G18876, AT5G17510, AT1G05370, AT4G21540, AT1 represented by any one of the polypeptide sequences selected G23900 and AT5G23690 (genes listed in Table A). Prefer from the group consisting of the polypeptides of Table A and ably, said AN3-based protein complex comprises at least the Tables A1 to A26. The overall sequence identity is determined proteins AN3p and one or more proteins selected from the using a global alignment algorithm, such as the Needleman group consisting of ARP4 (AT1G18450), ARP7 Wunsch algorithm in the program GAP (GCG Wisconsin (AT3G06830), SNF2 (AT2G46020), SYD (AT2G28290), Package. Accelrys), preferably with default parameters and SWI3C (AT1G21700) and SWP73B (AT5G14.170). Even preferably with sequences of mature proteins (i.e. without more preferably, said AN3-based protein complex comprises taking into account Secretion signals or transit peptides). at least AN3p. an actin related protein selected from the group Compared to overall sequence identity, the sequence identity consisting of ARP4 and ARP7, an ATPase selected from the will generally be higher when only conserved domains or group consisting of SNF2 (BRM) and SYD and a SWIRM motifs are considered. domain containing protein. Preferably, said SWIRM domain 0152 Preferably the methods of the invention concern a containing protein is SWI3C. An AN3-based protein complex homologue of an iSYT polypeptide derived from a crop plant as used here means that AN3p is interacting, directly or indi more preferably, in increasing order of preference from corn, rectly, with the other proteins of the complex. A direct inter Sugar cane, soybean, wheat, cotton and canola. action is an interaction where at least one domain of AN3p 0153. The terms “domain”. “signature” and “motif are interacts with one or more domains or the interaction partner. defined in the “definitions' section herein. An indirect interaction is an interaction where AN3p itself is 0154) In one embodiment preferred combinations of two not interacting with the interacting protein by one of its iSYT polypeptide whose expression is to be modulated domains, but where said interacting protein is interacting with according the methods of the invention are provided on Table a protein that is directly or indirectly interacting with AN3p. 3. In another further preferred embodiment homologues of 0157. A further aspect of the invention is the use of a such iSYT polypeptides are combined, more preferably from protein complex according to the invention to promote plant a monocotyledonous plant, more preferably from rice or corn growth. Preferably, said use is an overexpression of the pro plants. tein complex, by overexpressing at least two members of the TABLE 3 Combinations of two ISYT polypeptides. SYT SYT Combination polypeptide 1 ortholog of polypeptide 2 ortholog of Combi1 (AT5G28640) (AT1G 18450) Combi2 (AT5G28640) (AT3G60830) Combi3 (AT5G28640) (AT2G46020) Combia. (AT5G28640) (AT2G28290) Combi5 (AT5G28640) (AT1G21700) Combió (AT5G28640) (AT5G14.170) Combif (AT5G28640) (AT1G23900) Combi8 (AT5G28640) (AT2G18876) Combi9 (AT5G28640) (AT4G27550) Combi10 (AT5G28640) (AT1G65980) Combi11 (AT5G28640) (AT1G05370) Combi12 (AT5G28640) (AT4G35550) Combi13 (AT5G28640) (AT4G21540) Combi14 (AT5G28640) (AT1G20670) Combi15 (AT2G46020) (AT2G28290) Combi16 (AT5g23690) (AT3G60830) Combi17 (AT3G60830) (AT1G 18450) Combi18 (AT4g17330) (AT2G46020) Combi19 Poptr importin beta AT5G53480 Poptr importin alpha AT3g06720 Combi20 Zeama SYT1 ATSG28640 Zeama SWIBMDM2/CHC1 ATSG14170 like protein Combi21 Zeama SYT1 AT5G28.640 Zeama hypothetical ATSG-17510 protein, heme binding Combi22 Zeama SYT1 ATSG28640 Poptr ARP7 AT3G60830 Combi23 Poptr ARP7 AT3G60830 Poptr ARP4 AT1 G184SO

0155 Encompassed on Table 4 are the corresponding protein complex. Promotion of plant growth, as used here, is orthologoues iSYT-like polypeptides originating from poplar an increase in plant biomass in plants where the protein com or corn cells. plex is used, compared with the same plant where the com 0156 Another aspect of the invention is an isolated AN3 plex is not used, grown under the same conditions, except for based protein complex, comprising at least the proteins AN3p the conditions needed for the use of the complex, if any. Such and one or more of the proteins selected from the group conditions may be, as a non limited example, the addition of encoded by AT4G16143, AT1 G09270, AT3G06720, one or more compounds to induce one or more promoters of AT5G53480, AT3G60830, AT1 G18450, AT2G46020, one or more genes encoding a protein of the complex. Alter US 2015/0106974 A1 Apr. 16, 2015

natively, the same plant is an untransformed parental plant, 0164. The invention also provides hitherto unknown grown under the same conditions as the transformed plant, iSYT-encoding nucleic acids and iSYT polypeptides useful wherein the complex is used. Preferably, promotion of plant for conferring enhanced yield-related traits in plants relative growth results in an increased yield. This yield can be a total to control plants. increase in plant biomass, or a partial increase of yield, Such 0.165 According to a further embodiment of the present as, but not limited to seed yield, leave yield or root yield. invention, there is therefore provided an isolated nucleic acid molecule selected from: 0158 Still another aspect of the invention is a method to 0166 (i) a nucleic acid represented by any one of the promote AN3-based protein complex formation, by simulta polynucleotides of Tables A2 to A26; neous overexpression of at least two proteins of the complex. 0.167 (ii) the complement of a nucleic acid of (i); Proteins of the complex, beside AN3p itself, are listed in table 0168 (iii) a nucleic acid encoding an iSYT-like A. Preferably, said overexpression is an overexpression of polypeptide having in increasing order of preference at AN3p and one or more proteins selected from the group least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, consisting of ARP4 (AT1G 18450), ARP7 (AT3G60830). 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, SNF2 (AT2G46020), SYD (AT2G28290), SWI3C 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, (AT1G21700) and SWP73B (AT5G 14.170). Even more pref 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, erably, said overexpression is an overexpression of at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, AN3p, an actin related protein selected from the group con 95%.96%,97%.98% 99% or 100% sequence identity to sisting of ARP4 and ARP7, an ATPase selected from the the amino acid sequence of any of the polypeptides of group consisting of SNF2 (BRM) and SYD and a SWIRM Table A2 to A26 and further preferably conferring domain containing protein. Preferably, said SWIRM domain enhanced yield-related traits relative to control plants. containing protein is SWI3C. 0.169 (iv) a nucleic acid molecule which hybridizes 0159 Methods for obtaining overexpression are known to with a nucleic acid molecule of (i) to (iii) under high the person skilled in the art, and comprise, but are not limited stringency hybridization conditions and preferably con to placing the gene encoding the protein to be overexpressed fers enhanced yield-related traits relative to control after a strong promoter such as the Cauliflower Mosaic Virus plants. 35S promoter. Simultaneous overexpression as used here 0170 According to a further embodiment of the present means that there is an overlap in timeframe for all the proteins invention, there is also provided an isolated polypeptide to be overexpressed, whereby the level of said proteins is selected from: increased when compared to a non-overexpressed control. It 0171 (i) an amino acid sequence represented by any does not necessarily mean that all genes should be induced at one of the polypeptides of Tables A2 to A26; the same moment. Depending upon the turnover of the mes 0172 (ii) an amino acid sequence having, in increasing senger RNA and/or the protein, one gene may be induced order of preference, at least 50%, 51%, 52%. 53%, 54%, before or after another, as long as there is an overlap in time 55%, 56%, 57%, 58%, 59%, 60%, 61%. 62%, 63%, where both proteins are present in a concentration that is 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, higher than the normal (non-overexpressed) concentration. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 0160. In addition, two or three iSYT-like polypeptides, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% when expressed in rice according to the methods of the sequence identity to the amino acid sequence of any of present invention as outlined in the Examples section, give the sequences of the polypeptide of Table A2 to A26 and plants having increased yield related traits, selected from further preferably conferring enhanced yield-related increased aboveground biomass or increased seed yield. traits relative to control plants. 0161 The present invention is illustrated by transforming 0173 (iii) derivatives of any of the amino acid plants with the nucleic acid sequences comprising the gene sequences given in (i) or (ii) above. encoding the combinations of the polypeptides of Table 4. Nucleic acid variants may also be useful in practicing the methods of the invention. Examples of such variants include 0162. However, performance of the invention is not nucleic acids encoding homologues and derivatives of any restricted to these sequences; the methods of the invention one of the amino acid sequences given in Table A of the may advantageously be performed using any iSYT-like-en Examples section, the terms “homologue' and "derivative' coding nucleic acid or iSYT-like polypeptide as defined being as defined herein. Also useful in the methods of the herein. invention are nucleic acids encoding homologues and deriva 0163 Examples of nucleic acids encoding iSYT-like tives of orthologues or paralogues of any one of the amino polypeptides are given in Table Aan Tables A2 to A26 of the acid sequences given in Table A of the Examples section. Examples section herein. Such nucleic acids are useful in Homologues and derivatives useful in the methods of the performing the methods of the invention. The amino acid present invention have substantially the same biological and sequences given in Tables A2 to A26 of the Examples section functional activity as the unmodified protein from which they are example sequences of orthologues and paralogues of the are derived. Further variants useful in practising the methods iSYT polypeptide of Table A, the terms “orthologues' and of the invention are variants in which codon usage is opti “paralogues' being as defined herein. Further orthologues mised or in which miRNA target sites are removed. and paralogues may readily be identified by performing a 0.174 Further nucleic acid variants useful in practising the so-called reciprocal blast search as described in the defini methods of the invention include portions of nucleic acids tions section; where the query sequence is SEQID NO: 1 or encoding iSYT-like polypeptides, nucleic acids hybridising SEQID NO: 2, the second BLAST (back-BLAST) would be to nucleic acids encoding iSYT-like polypeptides, splice Vari against Arabidopsis thaliana sequences. ants of nucleic acids encoding iSYT-like polypeptides, allelic US 2015/0106974 A1 Apr. 16, 2015 22 variants of nucleic acids encoding iSYT-like polypeptides 0180. Another nucleic acid variant useful in the methods and variants of nucleic acids encoding iSYT-like polypep of the invention is a splice variant encoding a iSYT-LIKE tides obtained by gene shuffling. The terms hybridising polypeptide as defined hereinabove, a splice variant being as sequence, splice variant, allelic variant and gene shuffling are defined herein. as described herein. 0181. According to the present invention, there is provided 0175 Nucleic acids encoding iSYT-LIKE polypeptides a method for enhancing yield-related traits in plants, com need not be full-length nucleic acids, since performance of prising introducing and expressing in a plant a splice variant the methods of the invention does not rely on the use of of two or three nucleic acid sequences given in Table A or full-length nucleic acid sequences. According to the present Tables A2 to A26 of the Examples section, or a splice variant invention, there is provided a method for enhancing yield of a nucleic acid encoding an orthologue, paralogue or homo related traits in plants, comprising introducing and expressing logue of any of the amino acid sequences given in Table A of in a plant a portion of any one of the nucleic acid sequences the Examples section. given in Table A of the Examples section, or a portion of a 0182 Another nucleic acid variant useful in performing nucleic acid encoding an orthologue, paralogue or homo the methods of the invention is an allelic variant of a nucleic logue of any of the amino acid sequences given in Table A of acid encoding an iSYT-LIKE polypeptide as defined herein the Examples section wherein the nucleic acid encodes two or above, an allelic variant being as defined herein. three iSYT polypeptides. 0183. According to the present invention, there is provided 0176 A portion of a nucleic acid may be prepared, for a method for enhancing yield-related traits in plants, com example, by making one or more deletions to the nucleic acid. prising introducing and expressing in a plant of two or three The portions may be used in isolated form or they may be allelic variant of any one of the nucleic acids given in Table A fused to other coding (or non-coding) sequences in order to, or Tables A2 to A26 of the Examples section, or comprising for example, produce a protein that combines several activi Introducing and expressing in a plant an allelic variant of a ties. When fused to other coding sequences, the resultant nucleic acid encoding an orthologue, paralogue or homo polypeptide produced upon translation may be bigger than logue of any of the amino acid sequences given in Table A of that predicted for the protein portion. the Examples section. 0177 Portions useful in the methods of the invention, 0.184 Gene shuffling or directed evolution may also be encode a iSYT-like polypeptide as defined herein, and have used to generate variants of nucleic acids encoding iSYT Substantially the same biological activity as the amino acid LIKE polypeptides as defined above; the term “gene shuf sequences given in Table A of the Examples section. Prefer fling’ being as defined herein. ably, the portion is a portion of any one of the nucleic acids 0185. According to the present invention, there is provided given in Table A of the Examples section, or is a portion of a a method for enhancing yield-related traits in plants, com nucleic acid encoding an orthologue or paralogue of any one prising introducing and expressing in a plant a variant of any of the amino acid sequences given in Table A of the Examples one of the nucleic acid sequences given in Table A or Tables section. Preferably the portion is at least 100, 200, 300, 400, A2 to A26 of the Examples section, or comprising introduc 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 con ing and expressing in a plant a variant of a nucleic acid secutive nucleotides in length, the consecutive nucleotides encoding two or three orthologue, paralogue or homologue of being of any one of the nucleic acid sequences given in Table any of the amino acid sequences given in Table A or Tables A2 A of the Examples section, or of a nucleic acid encoding an to A26 of the Examples section, which variant nucleic acid is orthologue or paralogue of any one of the amino acid obtained by gene shuffling. sequences given in Table A of the Examples section. Most 0186. Furthermore, nucleic acid variants may also be preferably the portion is a portion of the nucleic acid of SEQ obtained by site-directed mutagenesis. Several methods are ID NO: 1. available to achieve site-directed mutagenesis, the most com 0178 Another nucleic acid variant useful in the methods mon being PCR based methods (Current Protocols in of the invention is a nucleic acid capable of hybridising, under Molecular Biology. Wiley Eds.). reduced stringency conditions, preferably under stringent 0187. Nucleic acids encoding iSYT-like polypeptides may conditions, with a nucleic acid encoding a iSYT-LIKE be derived from any natural or artificial source. The nucleic polypeptide as defined herein, or with a portion as defined acid may be modified from its native form in composition herein. and/or genomic environment through deliberate human 0179 Hybridising sequences useful in the methods of the manipulation. Preferably the iSYT-LIKE polypeptide-encod invention encode an iSYT-LIKE polypeptide as defined ing nucleic acid is from a plant, further preferably from a herein, having Substantially the same biological activity as monocotyledonous plant, most preferably the nucleic acid is the amino acid sequences given in Table A of the Examples from Zea mays or from Oryza sativa. section. Preferably, the hybridising sequence is callable of 0188 Performance of the methods of the invention gives hybridising to the complement of any one of the nucleic acids plants having enhanced yield-related traits. In particular per given in Table A of the Examples section, or to a portion of formance of the methods of the invention gives plants having any of these sequences, a portion being as defined above, or increased yield, especially increased seed yield relative to the hybridising sequence is capable of hybridising to the control plants. The terms yield' and “seed yield' are complement of a nucleic acid encoding an orthologue or described in more detail in the “definitions' section herein. paralogue of any one of the amino acid sequences given in 0189 Reference herein to enhanced yield-related traits is Table A of the Examples section. Most preferably, the hybri taken to mean an increase early vigour and/or in biomass dising sequence is capable of hybridising to the complement (weight) of one or more parts of a plant, which may include of a nucleic acid encoding any of the polypeptide of Table A aboveground (harvestable) parts and/or (harvestable) parts and Table A2 to A6. below ground. In particular, Such harvestable parts are seeds, US 2015/0106974 A1 Apr. 16, 2015

and performance of the methods of the invention results in enhanced yield-related traits relative to the parent plants, or to plants having increased seed yield relative to the seed yield of any other control plants as defined herein. control plants. 0200 Alternatively the nucleic acid sequences of (i), (ii) 0190. A preferred method for modulating (increasing or and (ii) are sequentially introduced and expressed by re decreasing) expression of any two or three nucleic acids transformation. Re-transformation is performed by introduc encoding the corresponding two or three iSYT-like polypep ing and expressing a nucleic acid sequence encoding one or tides is by introducing and expressing in a plant: (i) a nucleic two iSYT-like polypeptides, plant part, or plant cell compris acid sequence encoding a first iSYT-like polypeptide; and (ii) inga introduced and expressed nucleic acid sequence encod a nucleic acid sequence encoding a second iSYT-like ing a second one or two iSYT-like polypeptides, and prefer polypeptide. Therefore, according to the present invention, ably by selecting in the progeny for the presence and there is provided a method for enhancing yield-related traits expression of both transgenes. Therefore, according to the in plants, which method comprises introducing and express present invention, there is provided a method for enhancing ing in a plant: yield-related traits in plants, by re-transformation performed 0191 (i) any two or three nucleic acids encoding the by introducing and expressing a nucleic acid sequence encod corresponding two or three iSYT-like polypeptides; or ing one or two iSYT-like polypeptides into a plant, plant part, 0.192 (ii) two or three nucleic acids, each encoding a or plant cell comprising an introduced and expressed nucleic single iSYT-like polypeptide; or acid sequence encoding a second one or two iSYT-like 0193 (iii) a nucleic acid according to (i) and a nucleic polypeptide, and by preferably selecting in the progeny for according to (ii), the presence and expression of both transgenes, wherein said wherein said iSYT-like polypeptide is selected from the plants have enhanced yield-related traits relative to the plants group consisting of any of the polypeptides of Table A, homo having increased expression of one of logues thereof and fusions of the same. 0201 (i) any two or three nucleic acids encoding the 0194 Methods for introducing and expressing two or corresponding two or three iSYT-like polypeptides; or more transgenes (also called gene Stacking) in transgenic 0202 (ii) two or three nucleic acids, each encoding a plants are well known in the art (see for example, a review by single iSYT-like polypeptide; or Halpin (2005) Plant Biotech J (3): 141-155. Gene stacking can proceed by interactive steps, where two or more trans 0203 (iii) a nucleic acid according to (i) and a nucleic genes can be sequentially introduced into a plant by crossing according to (ii), a plant containing one transgene with individuals harbouring wherein said iSYT-like polypeptide is selected from the other transgenes or, alternatively, by re-transforming (or group consisting of any of the polypeptides of Table A, homo Super-transforming) a plant containing one transgene with logues thereof and fusions of the same. new genes. 0204 Alternatively, gene Stacking can occur via simulta 0.195 According to the present invention, there is provided neous transformation, or co-transformation, which is faster a method for enhancing yield-related traits in plants, which and can be used in a whole range of well known transforma method comprises sequentially introducing and expressing in tion techniques, preferably as described herein. a plant: 0205 Alternatively, gene Stacking can occur via simulta 0.196 (i) any two or three nucleic acids encoding the neous transformation, or co-transformation, which is faster corresponding two or three iSYT-like polypeptides; or and can be used in a whole range of transformation tech 0.197 (ii) two or three nucleic acids, each encoding a niques, as described in the “definition' section herein. single iSYT-like polypeptide; or 0206 When direct genetic transformation is considered, 0198 (iii) a nucleic acid according to (i) and a nucleic using physical or chemical delivery systems (e.g., micro according to (ii). projectile bombardment, PEG, electroporation, liposome, wherein said iSYT-like polypeptide is selected from the glass needles, etc.), the transgenes (at least two) can also be group consisting of any of the polypeptides of Table A, homo present in a number of conformations, but essentially do not logues thereof and fusions of the same. need to be comprised in a vector capable of being replicated 0199 Preferably, the nucleic acid sequences of (i), (ii) and in Agrobacteria or viruses, intermediates of the genetic trans (iii) are sequentially introduced and expressed by crossing. A formation. The two transgenes can be comprised in one or crossing is performed between a female parent plant compris more nucleic acid molecules, but simultaneously used for the ing an introduced and expressed isolated nucleic acid genetic transformation process. sequence encoding one or two iSYT-like polypeptides, and a 0207. According to the present invention, there is provided male parent plant also comprising an introduced and a method for enhancing yield-related traits in plants, which expressed isolated nucleic acid sequence encoding one or two method comprises simultaneously introducing and express iSYT-like polypeptides, and preferably selecting in the prog ing in a plant: (i) a nucleic acid sequence encoding one or two eny for the presence and expression of both transgenes. iSYT-like polypeptides; and (ii) a nucleic acid sequence Therefore, according to the present invention, there is pro encoding a second iSYT-like polypeptide, which plants have vided a method for enhancing yield-related traits in plants, by enhanced yield-related traits relative to plants having crossing a female parent plant comprising an introduced and increased expression of one of expressed isolated nucleic acid sequence encoding one or two iSYT-like polypeptides, and a male parent plant comprising 0208 (i) any two or three nucleic acids encoding the an introduced and expressed isolated nucleic acid sequence corresponding two or three iSYT-like polypeptides; or encoding one or two iSYT-like polypeptides, and preferably 0209 (ii) two or three nucleic acids, each encoding a selecting in the progeny for the presence and expression of at single iSYT-like polypeptide; or least two of the introduced transgenes encoding the corre 0210 (iii) a nucleic acid according to (i) and a nucleic sponding iSYT-like polypeptides, wherein said plants have according to (ii), US 2015/0106974 A1 Apr. 16, 2015 24 wherein said iSYT-like polypeptide is selected from the TABLE 4-continued group consisting of any of the polypeptides of Table A, homo logues thereof and fusions of the same. Preferred promoters 0211. The nucleic acid sequences of (i), (ii) and (iii) that are simultaneously introduced and expressed, are comprised SEQ ID in one or more nucleic acid molecules. Therefore, according Promoter name Source organism NO to the present invention is provided increasing yield-related ScBV Sugarcane bacilliform virus 667 traits in plants, which method comprises simultaneously SCBV-METI Sugarcane bacilliform virus 668 introducing and expressing in a plant with intron 0212 (i) any two or three nucleic acids encoding the ZnOBI Zea mayS 669 corresponding two or three iSYT-like polypeptides; or 0213 (ii) two or three nucleic acids, each encoding a 0223. In one preferred construct, a single control sequence single iSYT-like polypeptide; or is used to drive the expression of the nucleic acid sequence 0214 (iii) a nucleic acid according to (i) and a nucleic encoding two or three iSYT-like polypeptides, preferably according to (ii), those combinations as listed in Table 3. wherein said iSYT-like polypeptide is selected from the 0224. The present invention also provides for a mixture of group consisting of any of the polypeptides of Table A, homo constructs useful for example, for simultaneous introduction logues thereof and fusions of the same. and expression in plants of two or three nucleic acid sequence 0215. The invention also provides genetic constructs and encoding an iSYT-like polypeptideas defined above; wherein vectors to facilitate introduction and/or expression (de novo introduced or increased the already existing expression) in at least one construct comprises: plants of any two or three nucleic acids encoding the corre 0225 (a) a nucleic acid sequence nucleic acid sequence sponding combination of two or three iSYT-like polypep encoding an iSYT-like polypeptide as defined above: tides. The gene constructs may be inserted into vectors, which 0226 (b) one or more control sequences capable of may be commercially available, Suitable for transforming driving expression of the nucleic acid sequence of (a): into plants and for expression of the gene of interest in the and optionally transformed cells. The invention also provides use of a gene 0227 (c) a transcription termination sequence, construct as defined herein in the methods of the invention. and wherein at least one other construct comprises: 0216. More specifically, the present invention provides a 0228 (d) a nucleic acid sequence nucleic acid sequence construct comprising: encoding an iSYT-like polypeptide as defined above: 0217 (a) any two or three nucleic acids encoding the 0229 (e) one or more control sequences capable of corresponding two or three iSYT-like polypeptides as driving expression of the nucleic acid sequence of (d); defined above; and optionally 0218 (b) one or more control sequences capable of 0230 (f) a transcription termination sequence. increasing expression of the nucleic acid sequence of (a) 0231 Preferably, one of the control sequences of a con and of (b); and optionally struct is a constitutive promoter. An example of a constitutive 0219 (c) a transcription termination sequence. promoter is a GOS2 promoter, preferably a rice GOS2 pro 0220. The nucleic acid sequence of (a) is preferably a moter, more preferably a GOS2 promoter as represented by nucleic acid molecule comprising a nucleic acid sequence SEQID NO: 664 encoding combinations of two or three iSYT-like polypep 0232. The invention also provides for the use of a construct tides, preferably those combinations listed in Table 3. The comprising: (a) any two or three nucleic acids encoding the nucleic acid sequences encoding the iSYT polypeptides in corresponding two or three iSYT-like polypeptides as defined maybe fused to each other or separated by coding or non above, or of a mixture of constructs as described above, in a coding DNA. Such as promoters, introns, Subcellular target method for making plants having enhanced yield-related ing signal, or stuffed DNA such as the MARS (Matrix attach traits relative to plants having increased expression of one of ment Regions) regions. the nucleic acids encoding the corresponding two or three 0221) The term “control sequence” and “termination iSYT-like polypeptides which increased yield-related traits sequence' are as defined herein. Preferred control sequence are one or more of: (i) increased early vigour; (ii) increased of a construct useful in the methods of the invention are aboveground biomass or root biomass; (ii) increased total provided in Table 4, preferably as represented by SEQID NO: seed yield per plant; (iv) increased seed filling rate; (v) 665 to SEQID NO: 669. increased number of (filled) seeds; (vi) increased harvest 0222 Preferably, one of the control sequences of a con index; or (vii) increased thousand kernel weight (TKW). struct is a constitutive promoter. An example of a constitutive 0233. The invention also provides for plants, plant parts or promoter is a GOS2 promoter, preferably a rice GOS2 pro plant cells transformed with a construct comprising any two moter, more preferably a GOS2 promoter as represented by or three nucleic acids encoding the corresponding two or SEQID NO: 664. three iSYT-like polypeptides as defined above or with a mix ture of constructs as defined above. TABLE 4 0234 Plants are transformed with one or more vectors Preferred promoters comprising any of the nucleic acid sequences described above. The skilled artisan is well aware of the genetic ele SEQ ID ments that must be present on the vector in order to Success Promoter name Source organism NO fully transform, select and propagate host cells containing the GOS2 Oryza sativa 665 sequence of interest. The sequence of interest is operably HMGB Oryza sativa 666 linked to one or more control sequences (at least to a pro moter). US 2015/0106974 A1 Apr. 16, 2015

0235 Advantageously, any type of promoter, whether nucleic acid encoding a iSYT-Like polypeptide; however the natural or synthetic, may be used to increase expression of the effects of performing the method, i.e. enhancingyield-related nucleic acid sequence. A constitutive promoter is particularly traits may also be achieved using other well known tech useful in the methods. niques, including but not limited to T-DNA activation tag 0236. Other organ-specific promoters, for example for ging, TILLING, homologous recombination. A description preferred expression in leaves, stems, tubers, meristems, of these techniques is provided in the definitions section. seeds (embryo and/or endosperm), are useful in performing 0245. The present invention clearly extends to any plant the methods of the invention. See the “Definitions' section cellor plant produced by any of the methods described herein, herein for definitions of the various promoter types. and to all plant parts and propagules thereof. The present 0237. The present invention provides a method for invention encompasses plants or parts thereof (including enhancing yield-related traits especially seed yield of plants, seeds) obtainable by the methods according to the present relative to control plants, which method comprises modulat invention. The plants or parts thereof comprise a nucleic acid ing expression in a plant of a nucleic acid encoding a iSYT transgene encoding a iSYT-Like polypeptide as defined LIKE polypeptide as defined herein. above. The present invention extends further to encompass 0238 Since the transgenic plants according to the present the progeny of a primary transformed or transfected cell, invention have increased yield (yield related traits), it is likely tissue, organ or whole plant that has been produced by any of that these plants exhibit an increased growth rate (during at the aforementioned methods, the only requirement being that least part of their life cycle), relative to the growth rate of progeny exhibit the same genotypic and/or phenotypic char control plants at a corresponding stage in their life cycle. acteristic(s) as those produced by the parent in the methods 0239 According to a preferred feature of the present according to the invention. invention, performance of the methods of the invention gives 0246 The invention also includes host cells containing an plants having an increased growth rate relative to control isolated nucleic acid encoding an iSYT-like polypeptide as plants. Therefore, according to the present invention, there is defined hereinabove. Preferred host cells according to the provided a method for increasing the growth rate of plants, invention are plant cells. Host plants for the nucleic acids or which method comprises modulating expression in a plant of the vector used in the method according to the invention, the a nucleic acid encoding a iSYT-like polypeptide as defined expression cassette or construct or vector are, in principle, herein. advantageously all plants, which are capable of synthesizing 0240 Performance of the methods of the invention gives the polypeptides used in the inventive method. plants grown under non-stress conditions or under mild 0247 The methods of the invention are advantageously drought conditions increased yield relative to control plants applicable to any plant. Plants that are particularly useful in grown under comparable conditions. Therefore, according to the methods of the invention include all plants which belong the present invention, there is provided a method for increas to the Superfamily Viridiplantae, in particular monocotyle ingyield in plants grown under non-stress conditions or under donous and dicotyledonous plants including fodder or forage mild drought conditions, which method comprises modulat legumes, ornamental plants, food crops, trees or shrubs. ing expression in a plant of a nucleic acid encoding an iSYT According to a preferred embodiment of the present inven like polypeptide. tion, the plant is a crop plant. Examples of crop plants include 0241 Performance of the methods of the invention gives Soybean, Sunflower, canola, alfalfa, rapeseed, linseed, cotton, plants grown under conditions of nutrient deficiency, particu tomato, potato and tobacco. Further preferably, the plant is a larly under conditions of nitrogen deficiency, increased yield monocotyledonous plant. Examples of monocotyledonous relative to control plants grown under comparable conditions. plants include Sugarcane. More preferably the plant is a Therefore, according to the present invention, there is pro cereal. Examples of cereals include rice, maize, wheat, bar vided a method for increasing yield in plants grown under ley, millet, rye, triticale, Sorghum, emmer, spelt, secale, conditions of nutrient deficiency, which method comprises einkom, teff, milo and oats. modulating expressionina plant of a nucleic acid encoding an 0248. The invention also extends to harvestable parts of a iSYT-like polypeptide. plant such as, but not limited to seeds, leaves, fruits, flowers, 0242 Performance of the methods of the invention gives stems, roots, rhizomes, tubers and bulbs, which harvestable plants grown under conditions of salt stress, increased yield parts comprise a recombinant nucleic acid encoding an iSYT relative to control plants grown under comparable conditions. like polypeptide. The invention furthermore relates to prod Therefore, according to the present invention, there is pro ucts derived, preferably directly derived, from a harvestable vided a method for increasing yield in plants grown under part of such a plant, Such as dry pellets or powders, oil, fat and conditions of salt stress, which method comprises modulating fatty acids, starch or proteins. expression in a plant of a nucleic acid encoding an iSYT-Like 0249. The present invention also encompasses use of polypeptide. nucleic acids encoding iSYT-like polypeptides as described 0243 The invention also provides genetic constructs and herein and use of these iSYT-like polypeptides in enhancing vectors to facilitate introduction and/or expression in plants any of the aforementioned yield-related traits in plants. For of nucleic acids encoding iSYT-Like polypeptides. The gene example, nucleic acids encoding iSYT-like polypeptide constructs may be inserted into vectors, which may be com described herein, or the iSYT-like polypeptides themselves, mercially available, Suitable for transforming into plants and may find use inbreeding programmes in which a DNA marker suitable for expression of the gene of interest in the trans is identified which may be genetically linked to a iSYT-like formed cells. The invention also provides use of a gene con polypeptide-encoding gene. The nucleic acids/genes, or the struct as defined herein in the methods of the invention. iSYT-like polypeptides themselves may be used to define a 0244 As mentioned above, a preferred method for modu molecular marker. This DNA or protein marker may then be lating expression of a nucleic acid encoding a iSYT-Like used in breeding programmes to select plants having polypeptide is by introducing and expressing in a plant a enhanced yield-related traits as defined hereinabove in the US 2015/0106974 A1 Apr. 16, 2015 26 methods of the invention. Furthermore, allelic variants of a promoter, more preferably to a GOS2 promoter, most pref iSYT-like polypeptide-encoding nucleic acid/gene may find erably to a GOS2 promoter from rice. use in marker-assisted breeding programmes. Nucleic acids 0265 11. Method according to any one of items 1 to 10, encoding iSYT-like polypeptides may also be used as probes wherein said one or more said nucleic acids is of plant for genetically and physically mapping the genes that they are origin, preferably from a dicotyledonous plant, further a part of, and as markers for traits linked to those genes. Such preferably from the family Brassloaceae, more preferably information may be useful in plant breeding in order to from the genus Arabidopsis, most preferably from Arabi develop lines with desired phenotypes. dopsis thaliana. 0266 12. Plant or part thereof, including seeds, obtainable Items by a method according to any one of items 1 to 11, wherein 0250. The present invention will now be described in ref said plant or part thereof comprises any two or three erence to the embodiments of the following items: nucleic acids encoding the corresponding two or three 0251 1. A method for enhancing yield-related traits in a polypeptides selected from the group consisting of the plant relative to control plants, comprising modulating polypeptides listed in Table A, homologues thereof and expression in a plant of fusions of the same. 0252 (i) any two or three nucleic acids encoding the 0267 13. Construct comprising: corresponding two or three iSYT-like polypeptides; or 0268 (i) Any two or three nucleic acids encoding the 0253 (ii) two or three nucleic acids, each encoding a corresponding two or three polypeptides selected from single iSYT-like polypeptide; or the group consisting of any of the polypeptides listed in 0254 (iii) a nucleic acid according to (i) and a nucleic of Table A or homologues thereof and fusions of the according to (ii), Same: (0255 wherein said iSYT-like polypeptide is selected 0269 (ii) one or more control sequences capable of from the group consisting of any of the polypeptides of driving expression of the nucleic acid sequence of (i), Table A, homologues thereof and fusions of the same. preferably a plant promoter, more preferably a constitu 0256 2. A method according to item 1 wherein at least one tive promoter, even more preferably a GOS2 promoter, of the polypeptides is a synovial sarcoma translocation most preferably a GOS2 promoter from rice; and option (SYT) polypeptide or a homologue thereof, said SYT ally polypeptide or homologue thereof preferably comprising 0270 (iii) a transcription termination sequence. an SNH domain having in increasing order of preference at 0271 14. Construct according to item 12, wherein said least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, nucleic acid of (i) encodes two or three polypeptides 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, selected from the group consisting of the polypeptides 99% sequence identity to the SNH domain of SEQID NO: listed in Table 3. 670 (IQQYLDENKSLILKIVESQNSGKLSE CAENOARLQRNL MYLAAIAD). 0272. 15. Use of a construct according to item 13 or 14 in 0257 3. A method according to item 1 or 2 wherein said a method for making plants having increased yield, par nucleic acids encode the corresponding polypeptides ticularly increased biomass and/or increased seed yield Selected from the group consisting of the polypeptides relative to control plants. listed in Table 3. 0273 16. Plant, plant part or plant cell transformed with a 0258 4. Method according to any one of items 1 to 3, construct according to item 13 or 14. wherein said modulated expression is effected by introduc 0274 17. Method for the production of a transgenic plant ing and expressing in a plant said nucleic acids. having increased yield, particularly increased biomass 0259 5. Method according to any one of items 1 to 4, and/or increased seed yield relative to control plants, com wherein said nucleic acids is selected from the group con prising: sisting of the nucleic acids encoding any the proteins listed 0275 (i) introducing and expressing in a plant any two in Table A and Tables A2 to Table A26, or is a portion of or three nucleic acids encoding the corresponding Such a nucleic acid, or a nucleic acid capable of hybridising polypeptides selected from the group consisting of any with Such a nucleic acid. of the polypeptides of Table A or homologues thereof 0260 6. Method according to any one of items 1 to 5, and fusions of the same; and wherein said nucleic acids encode an orthologue or paral 0276 (ii) cultivating the plant cell underconditions pro ogue of any of the proteins given in Table A. moting plant growth and development. 0261 7. Method according to any preceding item, wherein 0277 18. Transgenic plant having increased yield, par said enhanced yield-related traits comprise increased ticularly increased biomass and/or increased seed yield, yield, preferably increased biomass and/or increased seed relative to control plants, resulting from modulated expres yield relative to control plants. sion of any two or three nucleic acids encoding the corre 0262 8. Method according to any preceding item, wherein sponding polypeptides selected from the group consisting said enhanced yield-related traits are obtained under non of any of the polypeptides of Table A or homologues stress conditions. thereof and fusions of the same, or a transgenic plant cell 0263 9. Method according to any preceding item, wherein derived from said transgenic plant. said enhanced yield-related traits are obtained under con 0278 19. Transgenic plant according to item 12, 16 or 18, ditions of drought stress, salt stress or nitrogen deficiency. or a transgenic plant cell derived thereof, wherein said 0264. 10. Method according to any one of items 4 to 9, plant is a crop plant or a monocot or a cereal. Such as rice, wherein said one or more said nucleic acids are operably maize, wheat, barley, millet, rye, triticale, sorghum emmer, linked to a plant promoter, preferably to a constitutive spelt, Secale, einkom, teff, milo and oats. US 2015/0106974 A1 Apr. 16, 2015 27

0279 20. Harvestable parts of a plant according to item 19, selected from the group consisting of ARP4 (AT1G 18450), wherein said harvestable parts are preferably shoot biom ARP7 (AT3G60830), SNF2 (AT2G46020), SYD asS and/or seeds. (AT2G28290), SWI3C (AT1G21700) and SWP73B 0280 21. Products derived from a plant according to item (AT5G 14.170). 18 or 19 and/or from harvestable parts of a plant according 0294 33. An isolated AN3-based protein complex accord to item 20. ing to item 2, whereby said protein complex comprises at 0281 22. Construct according to item 12, wherein said least AN3p, an actin related protein selected from the group nucleic acid of (i) encodes two polypeptides selected from consisting of ARP4 and ARP7, an ATPase selected from the group consisting of the combinations of Table 3. the group consisting of SNF2 (BRM) and SYD and a 0282) 23. Use of a construct according to item 13 or 14 in SWIRM domain containing protein. a method for making plants having increased yield, par 0295 34. An isolated AN3-based protein complex accord ticularly increased biomass and/or increased seed yield ing to item 3, whereby said SWIRM domain containing relative to control plants. protein is SWI3C 0283 24. Plant, plant part or plant cell transformed with a 0296 35. The use of a protein complex according to any of construct according to item 13 or 14. the preceding items to promote plant growth. 0284. 25. Method for the production of a transgenic plant 0297 36. A method to promote AN3-based protein com having increased yield, particularly increased biomass plex formation by simultaneous overexpression of at least and/or increased seed yield relative to control plants, com two proteins of the complex. prising: 0285 (iii) introducing and expressing in a plant one or DESCRIPTION OF FIGURES more (isolated) nucleic acids encoding at least one, pref erably two, three, four, five, six, seven, eight, nine, ten or 0298. The present invention will now be described with more polypeptides selected from the group consisting of reference to the following figures in which: any of the polypeptides of Table A, homologues thereof 0299 FIG. 1. Expression analysis of GS-tagged GFP and and fusions of the same; and AN3 in transgenic cell Suspension cultures. 0286 (iv) cultivating the plant cell under conditions 0300. The total protein extract of 2-day-old wild-type and promoting plant growth and development. N- and C-terminal GS-tagged GFP and AN3 overexpressing 0287. 26. Transgenic plant having increased yield, par cultures (60 ug) was separated by 12% SDS-PAGE and ticularly increased biomass and/or increased seed yield, immunoblotted. relative to control plants, resulting from modulated expres 0301 For detection of GS-tagged proteins, blots were sion of one or more (isolated) nucleic acids encoding at incubated with human blood plasma followed by incubation least one, preferably two, three, four, five, six, seven, eight, with anti-human IgG coupled to horseradish peroxidase. Pro nine, ten or more polypeptides selected from the group tein gel blots were developed by Chemiluminiscent detection. consisting of any of the polypeptides of Table A, homo The expected recombinant molecular masses for GS-tagged logues thereof and fusions of the same, or a transgenic GFP and AN3 are 52.8 kDa and 43.5 kDa, respectively (indi plant cell derived from said transgenic plant. cated with a black dot). 0288. 27. Transgenic plant according to item 12, 16 or 18, (0302 FIG. 2. Analysis of the TAP protein eluates. or a transgenic plant cell derived thereof, wherein said 0303 GS-tagged protein complexes were purified from plant is a crop plant or a monocot or a cereal. Such as rice, transgenic plant cell Suspension cultures, precipitated with maize, wheat, barley, millet, rye, triticale, sorghum emmer, TCA (25%, V/v), separated on 4-12% NuPAGE gels, and spelt, Secale, einkom, teff, milo and oats. visualized with colloidal Coomassie G-250 staining. Bait 0289 28. Harvestable parts of a plant according to item 19, proteins are indicated with a dot. wherein said harvestable parts are preferably shoot biom 0304 FIG. 3. represents the binary vector used for intro asS and/or seeds. ducing and expressing in Oryza sativa of a iSYT-Like-encod 0290 29. Products derived from a plant according to item ing nucleic acid under the control of a plant promoter. 18 or 19 and/or from harvestable parts of a plant according to item 20. EXAMPLES 0291 30. Use of any two or three nucleic acids encoding two or three polypeptides selected from the group consist (0305. The present invention will now be described with ing of any of the polypeptides of Table A, homologues reference to the following examples, which are by way of thereof and fusions of the same in increasing yield, par illustration alone. The following examples are not intended to ticularly in increasing seed yield and/or shoot biomass in completely define or otherwise limit the scope of the inven plants, relative to control plants. tion. 0292) 31. An isolated AN3-based protein complex, com 0306 DNA manipulation: unless otherwise stated, recom prising at least the proteins AN3p and one or more of the binant DNA techniques are performed according to standard proteins selected from the group encoded by AT4G16143. protocols described in (Sambrook (2001) Molecular Cloning: AT1G09270, AT3G06720, AT5G53480, AT3G60830. a laboratory manual, 3rd Edition Cold Spring Harbor Labo AT1 G18450, AT2G46020, AT2G28290, AT1G21700, ratory Press, CSH, New York) or in Volumes 1 and 2 of AT5G14170, AT4G17330, AT4G27550, AT1G65980, Ausubel et al. (1994), Current Protocols in Molecular Biol AT5G55210, AT3G 15000, AT4G35550, AT1G20670, ogy, Current Protocols. Standard materials and methods for AT1G08730, AT5G13030, AT2G18876, AT5G17510, plant molecular work are described in Plant Molecular Biol AT1G05370, AT4G21540, AT1G23900 and AT5G23690. ogy Labfax (1993) by R. D. D. Croy, published by BIOS 0293 32. An isolated AN3-based protein complex com Scientific Publications Ltd (UK) and Blackwell Scientific prises at least the proteins AN3p and one or more proteins Publications (UK). US 2015/0106974 A1 Apr. 16, 2015 28

Example 1 Example 4 Vector Construction: TAP Vectors Expression Analysis of Cell Suspension Cultures 0307 Construction of N- and C-terminal GS-tagged GFP 0311 Transgene expression was analyzed in a total pro and AN3 under the control of the 35S (CaMV) promoter was tein extract derived from exponentially growing cells, har obtained by Multisite Gateway LR reactions. The coding Vested two days after Subculturing. Equal amounts of total regions, without (-) and with (+) stopcodon, were amplified protein were separated on 12% SDS-PAGE gels and blotted by polymerase chain reaction (PCR) and cloned into the onto Immobilon-P membranes (Millipore, Bedford, Mass.). Gateway p)ONR221 vector (Invitrogen) resulting in Protein gel blots were blocked in 3% skim milk in 20 mM pEntryL1 L2-GFP(-), pFntryL1L2-GFP(+), pFntryL1L2 Tris-HCl, pH 7.4, 150 mMNaCl, and 0.1% TritonX-100. For AN3(-) and pEntryL1L2-AN3(+). The Pros:GFP-GS- and detection of GS-tagged proteins, blots were incubated with Pross:AN3-GS-containing plant transformation vectors human blood plasma followed by incubation with anti-human were obtained by Multisite Gateway LR reaction between IgG coupled to horseradish peroxidase (HRP; GE-Health pEntryL4R1-Pro, pEntryL1L2-GFP(-) or pEntryL1L2-AN3 care). Protein gel blots were developed by Chemiluminiscent (-), and pEntry R2L3-GS and the destination vectorpKCTAP, detection (Perkin Elmer, Norwalk, Conn.). respectively (Van Leene et al., 2007). To obtain the Pro35S: GS-GFP and Pro35S:GS-AN3 vectors Multisite LR recom Example 5 bination between pentryL4L3-Pross and pEntryL1L2-GFP (+) or pEntryL1L2-AN3(+) with pKNGSTAP occurred. Protein Extract Preparation 0308 All entry and destination vectors were checked by sequence analysis. Expression vectors were transformed to 0312 Cell material (15g) was grinded to homogeneity in Agrobacterium tumefaciens strain C58C1Rif (pMP90) by liquid nitrogen. Crude protein extract were prepared in an electroporation. Transformed bacteria were selected on yeast equal volume (w/v) of extraction buffer (25 mM Tris-HCl, pH extract broth plates containing 100 g/mL rifampicin, 40 7.6, 15 mM MgCl, 5 mM EGTA, 150 mM NaCl, 15 mM ug/mL gentamicin, and 100 ug/mL spectinomycin. p-nitrophenylphosphate, 60 mM f-glycerophosphate, 0.1% (v/v) Nonidet P-40 (NP-40), 0.1 mM sodium vanadate, 1 mM NaF. 1 mM DTT, 1 mM PMSF, 10 g/mL leupeptin, 10 Example 2 ug/mL aprotinin, 5ug/mL antipain, 5ug/mL chymostatin, 5 ug/mL pepstatin, 10 ug/mL Soybean trypsin inhibitor, 0.1 Cell Suspension Cultivation mMbenzamidine, 1 M trans-epoxysuccinyl-L-leucylamido 0309 Wild-type and transgenic Arabidopsis thaliana cell (4-guanidino)butane (E64), 5% (v/v) ethylene glycol) using suspension PSB-D cultures were maintained in 50 mL an Ultra-Turrax T25 mixer (IKA Works, Wilmington, N.C.) at MSMO medium (4.43 g/L MSMO, Sigma-Aldrich), 30 g/L 4°C. The soluble protein fraction was obtained by a two-step sucrose, 0.5 mg/L NAA, 0.05 mg/L kinetin, pH 5.7 adjusted centrifugation at 36900 g for 20 min and at 178000 g for 45 with 1MKOH) at 25°C. in the dark, by gentle agitation (130 min, at 4°C. The extract was passed through a 0.45 um filter rpm). Every 7 days the cells were subcultured in fresh (Alltech, Deerfield, Ill.) and the protein content was deter medium at a 1/10 dilution. mined with the Protein Assay kit (Bio-Rad, Hercules, Calif.). Example 3 Example 6 Cell Culture Transformation Tandem Affinity Purification 0310. The Arabidopsis culture was transformed by Agro 0313 Purifications were performed as described by bacterium co-cultivation as described previously (Van Leene Birckstimmer et al. (2006), with some modifications. et al., 2007). The Agrobacterium culture exponentially grow Briefly, 200 mg total protein extract was incubated for 1 h at ing in YEB (ODoo between 1.0 and 1.5) was washed three 4°C. under gentle rotation with 100 uL IgG Sepharose 6 Fast times by centrifugation (10 min at 5000 rpm) with an equal Flow Flow beads (GE-Healthcare, Little Chalfont, UK), pre volume MSMO medium and resuspended in cell suspension equilibrated with 3 mL extraction buffer. The IgG Sepharose growing medium until an ODoo of 1.0. Two days after Sub beads were transferred to a 1 mL Mobicol column (MoBiTec, cultivation, 3 mL suspension culture was incubated with 200 Goettingen, Germany) and washed with 10 mL IgG wash uL washed Agrobacteria and 200 uMacetoseringone, for 48 buffer (10 mM Tris-HCl, pH 8.0, 150mMNaCl, 0.1% NP-40, h in the dark at 25°C. with gentle agitation (130 rpm). Two 5% ethylene glycol) and 5 mL Tobacco (Nicotiana tabacum days after co-cultivation, 7 mL MSMO containing a mix of L) Etch Virus (TEV) buffer (10 mM Tris-HCl, pH 8.0, 150 three antibiotics (25 ug/mL, kanamycin, 500 g/mL carbeni mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM PMSF, cellin, and 500 ug/mL. Vancomycin) was added to the cell 1 ME64, 5% (v/v) ethylene glycol). Bound complexes were cultures and grown further in Suspension understandard con eluted via AcTEV digest (2x100 U, Invitrogen) for 1 h at 16° ditions (25°C., 130 rpm and continuous darkness). The stable C. The IgG eluted fraction was incubated for 1 hat 4°C. under transgenic cultures were selected by sequential dilution in a gentle rotation with 100LL Streptavidin resin (Stratagene, La 1:5 and 1:10 ratio in 50 mL fresh MSMO medium containing Jolla, Calif.), pre-equilibrated with 3 mL TEV buffer. The the antibiotics mix, respectively at 11, and 18 days post co Streptavidin beads were packed in a Mobicol column, and cultivation. After counter selecting the bacteria, the trans washed with 10 mL TEV buffer. Bound complexes were genic plant cells were further subcultured weekly in a 1:5 eluted with 1 mL streptavidin elution buffer (10 mM Tris ratio in 50 mL MSMO medium containing 25 g/mL kana HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM mycin for two more weeks. Thereafter the cells were weekly EDTA, 1 mM PMSF, 1 uM E64, 5%(v/v) ethylene glycol, 20 subcultured in fresh medium at a 1/10 dilution. mM Desthiobiotin), and precipitated using TCA (25% V/v). US 2015/0106974 A1 Apr. 16, 2015 29

The protein pellet was washed twice with ice-cold aceton Data search files were generated and submitted for protein containing 50 mM HCl, redissolved in sample buffer and homology identification by using a local database search separated on 4-12% gradient NuPAGEgels (Invitrogen). Pro engine (Mascot 2.1, Matrix Science). An in-house nonredun teins were visualized with colloidal Coomassie brilliant blue dant Arabidopsis protein database called SNAPS Arabidopsis staining. thaliana version 0.4 (SNAPS-Simple Nonredundant Assem bly of Protein Sequences, 77488 sequence entries, 30468560 Example 7 residues; available at ptools.ua.ac.be/snaps) was compiled from nine public databases. Proteinhomology identifications Proteolysis and Peptide Isolation of the top hit (first rank) with a relative score exceeding 95% 0314. After destaining, gel slabs were washed for 1 hour in probability were retained. Additional positive identifications H2O, polypeptide disulfide bridges were reduced for 40 min (second rank and more) were retained when the score in 25 mL of 6.66 mM DTT in 50 mMNHHCO, and sequen exceeded the 98% probability threshold. tially the thiol groups were alkylated for 30 min in 25 mL 55 mMIAM in 50 mMNHHCO. Afterwashing the gel slabs 3 Example 10 times with water, complete lanes from the protein gels were Expression Analysis of GS-Tagged GFP and AN3 cut into slices, collected in microtiterplates and treated essen Overexpressing Cell Lies tially as described before with minor modifications (Van Leene et al., 2007). Per microtiter plate well, dehydrated gel 0317 Before performing TAP purifications stably trans particles were rehydrated in 20 uL digest buffer containing formed cell Suspension cultures were screened on the protein 250 ng trypsin (MS Gold; Promega, Madison, Wis.), 50 mM expression level of the transgenes. Protein gel blotting of NHHCO, and 10% CHCN (v/v) for 30 min at 4°C. After equal amounts of total protein extract derived from wild-type adding 10 uIl of a buffer containing 50 mM NHHCO, and (PSB-D) cultures and GS-GFP, GFP-GS, GS-AN3, and AN3 10% CHCN (v/v), proteins were digested at 37° C. for 3 GS overexpressing cell lines showed clear expression of the hours. The resulting peptides were concentrated and desalted GS-tagged proteins (FIG. 1). with microcolumn solid phase tips (PerfectPureTM C18 tip, 200 n bed volume: Eppendorf, Hamburg, Germany) and Example 11 eluted directly onto a MALDI target plate (Opti-TOFTM384 Well Insert Applied Biosystems, Foster City, Calif.) using 1.2 TAP Purification of Wild-Type and GS-Tagged GFP uL of 50% CHCN: 0.1% CF,COOH solution saturated with Overexpress AN3 Cultures C-cyano-4-hydroxycinnamic acid and spiked with 20 fmole? uL Glu1-Fibrinopeptide B (Sigma-Aldrich), 20 fmolefulL 0318 Despite the two successive purification steps per des-Pro2-Bradykinin (Sigma-Aldrich), and 20 fmolefulL formed within TAP purifications, background proteins co Adrenocorticotropic Hormone Fragment 18-39 human purified by non-specific binding are an issue. Contaminating (Sigma-Aldrich). proteins due to experimental background were determined by purifications on wild-type and transgenic cultures overex Example 8 pressing N- and C-terminal GS-tagged nuclear localized green fluorescent protein (GFP). Non-specific co-purified Acquisition of Mass Spectra proteins were precipitated, separated on gel, stained (FIG. 2), trypsin digested and identified unambiguously by MALDI 0315. A MALDI-tandem MS instrument (4800 Proteom TOF/TOF. Most contaminants are high abundant proteins, ics Analyzer, Applied BioSystems) was used to acquire pep Such as chaperones, cytoskeleton proteins, ribosomal pro tide mass fingerprints and Subsequent 1 kV CID fragmenta teins, metabolic enzymes, or protein translation factors tion spectra of selected peptides. Peptide mass spectra and (Table 0). Identical or similar proteins were found as common peptide sequence spectra were obtained using the settings contaminants in other plant protein-protein interaction stud essentially as presented in Van Leene et al. (2007). Each ies (Rohila et al., 2006; Van Leone et al., 2007). MALDI plate was calibrated according to the manufacturers specifications. All peptide mass fingerprinting (PMF) spectra Example 12 were internally calibrated with three internal standards at m/z. 963.516 (des-Pro2-Bradykinin), m/z 1570.677 (Glu1-Fibrin TAPIsolation and MS Identification of AN3 opeptide B), and m/z. 2465,198 (Adrenocorticotropic Hor Interacting Proteins mone Fragment 18-39) resulting in an average mass accuracy of 5 ppm-t10 ppm for each analyzed peptide spot on the 0319. In order to identify the interaction partners of AN3 analyzed MALDI targets. Using the Individual PMF spectra, in vivo, we performed tandem affinity (TAP) purifications on up to sixteen peptides, exceeding a signal-to-noise ratio of 20 N- and C-terminal GS-fusions of AN3 ectopically expressed that passed through a mass exclusion filter were Submitted to under control of the constitutive 35SCaMV promoter intrans fragmentation analysis. genic Arabidopsis Suspension cultures. Two independent TAP purifications were performed on extracts from AN3-GS Example 9 and GS-AN3 lines, harvested two days after sub-culturing into fresh medium. The affinity purified proteins were sepa rated on a 4-12% NuPAGE gel and stained with Coomassie MS-Based Protein Homology Identification Brilliant Blue. The purification profiles from transgenic cul 0316 PMF spectra and the peptide sequence spectra of tures overexpressing AN3 is shown in FIG. 2. Protein bands each sample were processed using the accompanied Software were cut, in-gel digested with trypsin and Subjected to suite (GPS Explorer 3.6, Applied Biosystems) with parameter MALDI-TOF/TOF mass spectrometry for protein identifica settings essentially as described in Van Leone et al. (2007). tion. After Subtracting background proteins, identified by the US 2015/0106974 A1 Apr. 16, 2015 30 control purifications described in example 2 and in other ity (Kandasamy et al., 2005a). ARP7 knockdown results in analyses (GUS and cytosolic GFP. Van Leene et al., 2007), dwarfed plants with small rosette leaves, highly retarded root from the obtained hit list we identified 25 AN3 Interacting growth, altered flower development and reduced fertility proteins (Table A). These can be divided into two groups: 14 (Kandasamy et al., 2005b). Finally, RNAi-mediated silencing proteins were confirmed experimentally and 11 proteins were of the accessory SWI/SNF complex component SWP73B identified only in one out of four TAP experiments. (At5g 14.170) resulted in dwarfed plants with shorter roots (Crane & Gelvin, 2007). Example 13 Example 14 Isolation and Subunit Identification of AN3 Interacting SWI/SNFChromatin Remodeling Isolation and Identification of AN3 Interactors Complexes in Plants 0323 With the exception of the SWI/SNF chromatin remodeling complex subunits all other 19 identified AN3 0320 Among the experimentally confirmed AN3 interac interactors are not or poorly characterized. Table B gives an tors six proteins act as Subunits of macromolecular machines overview of their GO biological process and molecular func that remodel chromatin structure. A database Survey tion. (ChromDB, Gendler et al., 2008) Illustrates that all of them 0324. Among them four interactors (Atag16143. belong to the SWI/SNFATPase family. SWI/SNF chromatin At1g09270, At3g06720 and At5g53480) are involved in remodeling ATPases are conserved in the animal and the plant nucleocytoplasmic trafficking which identifies AN3 as one of kingdom and regulate transcriptional programs in response to the targets of plant nuclear transporters. Indeed a precise endogenous and exogenous cues. This suggests that the tran cellular localization is essential for protein function and scriptional activity of AN3 is regulated through chromatin nuclear localization is a key to the function of transcription remodeling. In agreement, the human AN3 homolog SYT factors. In plants, nucleocytoplasmic trafficking plays a criti was also shown to interact with the SWI/SNF complex com cal role in various biological processes (Meier, 2007; Xu & ponents BRM and Brg1 (Thaete et al., 1999; Perani at al., Meier, 2008) and nuclear transporters have been shown to be 2003: Ishida et al., 2004). involved in regulating different signal transduction pathways 0321 Although the functional role of several putative during plant development (Bollman et al., 2003) and in plant SWI/SNF complex components has been studied in Arabi responses to biotic (Palma et al., 2005) and abiotic stresses dopsis, so far no complete plant chromatin remodeling com (Verslues et al., 2006). plex has been isolated and characterized. The co-purification 0325 Another AN3 interactor, that is yet not character with AN3 gives for the first time proof of the in vivo physical ized, is the trehalose phosphatase/synthase 4 (TPS4). Several composition of plant SWI/SNF complexes which before was studies in plants imply an important role of trehalose biosyn based solely on homology analyses and the interpretation of thesis for plant growth, development and stress tolerance genetic and in vitro interactions. (Grennan, 2007). In the case of Arabidopsis TPS1, knockout 0322. A literature survey illustrates that SWI/SNFATPase mutants display an embryo lethal phenotype, Suggesting a Subunits control multiple developmental pathways in Arabi role of this gene in plant development (Eastmondet al., 2002). dopsis. Null mutants of the two isolated ATPases SYD In addition, overexpression of TPS1 shed light on its role as a (At2g28290) and BRM (SNF2) (At2g46020) display pleio regulator of glucose, abscisic acid, and stress signalling tropic developmental defects. Both mutants are slow growing (Avonce et al., 2004). The latter study, together with a recent and dwarfed, have defects in cotyledon separation, and analysis of a rice TPS triggering abiotic stress response gene exhibit reduced apical dominance (Wagner & Meyerowitz, induction when overexpressed (Geet al., 2008), Suggests a 2002; Farrona et al., 2004; Hurtado et al., 2006; Kwon et al., possible role for TPS genes in regulating transcriptional sig 2006; Suet al., 2006). Null mutants in BRM (SNF2) also have naling pathways. unique root growth defects and are male Sterile (Wagner & 0326. The other identified interactors indicate links of Meyerowitz, 2002; Hurtado et al., 2006; Kwon et al., 2006). AN3 function in multiple processes. Several studies demon Core complex Swi3c (Atlg21700) mutants closely resemble strate the involvement of sphingosine kinases in plant cell brm mutants (Sarnowski et al., 2005). Mutants of the acces signaling (Coursolet al., 2003: Coursolet al., 2005: Worral et sory components ARP4 and ARP7 display pleiotropic defects al., 2008), whereas reports on myosinhomologues (Peremys with less resemblance to the syd, brm and Swi3c phenotypes lov et al., 2008; Jiang et al., 2007) implicate roles of protein (Meagher et al., 2005). Down-regulation of ARP4 resulted in and organelle trafficking in plant development. The connec phenotypes including altered organization of plant organs, tions between these genes, the other identified interactors and early flowering, delayed flower senescence and partial steril AN3 will be interesting to study in the future. TABLE O List of co-purifying proteins during TAP experiments of untransformed cell cultures, and of cultures ectopically expressing nuclear localized GFP Accession number Protein name Mock GFP At1g06780 glycosyltransferase family 8 protein -- At1g07930 elongation factor 1-alpha -- At1g09080 luminal binding protein 3 (BiP3) (BP3) -- Atlg13440 glyceraldehyde 3-phosphate dehydrogenase, cytosolic, -- Atlg31230 bifunctional aspartate kinase/homoserine dehydrogenase -- US 2015/0106974 A1 Apr. 16, 2015 31

TABLE O-continued List of co-purifying proteins during TAP experiments of untransformed cell cultures, and of cultures ectopically expressing nuclear localized GFP Accession number Protein name Mock GFP Atlg34610 Ulp1 protease family protein -- Atlg50010 tubulin alpha chain -- Atlg61210 WD-40 repeat family protein katanin p80 subunit, putative -- Atlg75010 MORN repeat-containing protein -- Atlg79920 heat shock protein 70, putative -- Atlg79930 heat shock protein, putative -- At2g07620 putative helicase -- At2g21410 vacuolar proton ATPase, putative -- At2g26570 expressed protein -- At3g07160 glycosyltransferase family 48 protein -- At3g09170 Ulp1 protease family protein -- At3g09440 heat shock cognate 70 kDa protein 3 -- At3g11950 ATHST: prenyltransferase -- At3g12580 heat shock protein 70, putative -- At3g17390 S-adenosylmethionine synthetase, putative -- At3g18530 expressed protein -- At3g26020 serine/threonine protein phosphatase 2A regulatory subunit B' -- At3.g42100 AT hook motif-containing protein-related -- At3.g48870 ATP-dependent Clp protease ATP-binding subunit (ClpC) -- At3g49640 nitrogen regulation family protein -- At3g54940 cysteine proteinase, putative -- At4g.00020 BRCA2A (breast cancer 2 like 2A) -- At4g.09800 40S ribosomal protein S18 -- At4g14960 tubulin alpha chain -- At4g18080 hypothetical protein -- At4g20160 expressed protein -- At4g20890 tubulin beta chain -- At4.g31820 phototropic-responsive NPH3 family protein -- At4.g33200 myosin, putative -- At 5g02490 heat shock cognate 70 kDa protein 2 -- At 5g02500 heat shock cognate 70 kDa protein 1 -- At 5g08670 ATP synthase beta chain, mitochondrial -- At 5g08680 ATP synthase beta chain, mitochondrial -- At 5g08690 ATP synthase beta chain, mitochondrial -- At5g.09810 actin 7 (ACT7)/actin 2 -- -- At 5g18110 Novel cap-binding protein (nGBP) -- At 5g285.40 luminal binding protein 1 (BiP-1) (BP1) -- -- At 5.g35360 acetyl-CoA carboxylase, biotin carboxylase subunit (CAC2) -- At 5g40060 disease resistance protein (TIR-NBS-LRR class), putative -- At 5g42020 luminal binding protein 2 (BiP-2) (BP2) -- At 5g44340 tubulin beta chain -- At 5g60390 elongation factor 1-alpha -- At 5g62700 tubulin beta chain --

TABLE A AN3 and List of AN3-copurified proteins identified by MS. The last column tells in how many of the four independent experiments an interactor was identified. Peptide Protein Bestion AGI code Description MW (kDa) count score/threshold score/threshold ATSG28640 AN3, SYT1, SYT, GIF1 AT4G16143 importin alpha-2, putative (IMPA2) 49.5 13 388,61 84.28 2 AT1G09270 importin alpha-1 subunit, putative (IMPA4) 59.4 6 74f61 37/31 1 AT3GO6720 importin alpha-1 subunit, putative (IMPA1) 58.6 8 16061 62.28 2 AT5G53480 importin beta-2, putative 96.2 16 295,61 50.32 2 AT3G60830 actin-related protein 7 (ARP7) 39.9 12 285,61 53/28 3 AT1G18450 actin-related protein 4 (ARP4) 48.9 12 230,61 44,28 2 AT2G46020 transcription regulatory protein SNF2 245.4 31 351,61 57.31 2 (ATPase) AT2G28290 chromatin remodeling protein, SYDATPase 389.8 22 11861 53.31 4 AT1G21700 SWIRM domain-containing protein DNA- 88.2 5 32.32 2 binding family protein AT5G14170 SWIB complex BAF60b domain-containing 59.2 18 3O2.61 43.31 2 protein AT4G17330 G2484-1, agenet (tudor-like) domain- 113.3 25 317,61 61.32 3 containing protein US 2015/0106974 A1 Apr. 16, 2015 32

TABLE A-continued AN3 and List of AN3-copurified proteins identified by MS. The last column tells in how many of the four independent experiments an interactor was identified. Peptide Protein Bestion AGI code Description MW (kDa) count score/threshold score/threshold AT4G27550 trehalose phosphatase/synthase 4 89.4 15 68.6 2 AT1G65980 thioredoxin-dependent peroxidase 17.4 8 80.6 2 AT5G55210 expressed protein 18.5 4 105.6 49.31 2 AT3G15000 expressed protein similar to DAG protein 42.8 3 38.30 2 AT4G35550 homeobox-leucine zipper protein (HB-2), 29.6 3 33,28 1 HD-ZIP protein AT1G20670 DNA-binding bromodomain-containing 72.9 16 75/6 1 protein AT1G08730 myosin heavy chain (PCR43) (Fragment) 1746 18 70,6 1 AT5G13030 expressed protein 71.1 3 31.29 1 AT2G18876 expressed protein 43.5 11 67.6 1 AT5G17510 expressed protein 42.5 3 37,28 1 AT1G05370 expressed protein 49.9 12 666 1 AT4G21540 putative sphingosine kinase (SphK) 1417 9 69.6 1 AT1G23900 gamma-adaptin 96.4 19 78.6 1 AT5G23690 polynucleotide adenylyltransferase family 59.6 11 666 1 protein

TABLE B

GO Molecular AGI Code Name/Description GO Biological Process Function At4g16143 Importin alpha-2 Protein import into nucleus Protein transporter (IMP2) activity Atlg09270 Importin alpha-1 Intracellular protein Protein transporter (IMPA4) transport activity At3g06720 Importin alpha-1 Intracellular protein Protein transporter (IMPA1) transport activity At 5g53480 Importin beta-2 Protein import into nucleus Protein transporter activity At4g17330 G2484-1 protein unknown RNA binding AtAg27550 Trehalose Trehalose biosynthesis Trehalose phosphate phosphatase, synthase synthase activity 4 (TPS4) Atlg65980 Thioredoxin- unknown Antioxidant activity dependent peroxidase (TPX1) At 5g55210 Expressed protein unknown unknown At3g15000 Expressed protein unknown unknown similar to DAG protein AtAg35550 Wuschel-related Regulation of transcription DNA binding homeobox 13 (WOX13) Atlg20670 Bromodomain- unknown DNA binding containing protein Atlgo8730 Myosin-like protein Actin filament-based Protein binding XIC movement At 5g13030 Expressed protein unknown unknown At2g18876 Expressed protein unknown unknown At 5g17510 Expressed protein unknown unknown Atlgo5370 Expressed protein unknown unknown At4g21540 Putative sphingosine Activation of protein kinase Kinase activity kinase C activity Atlg23900 Gamma-adaptin Vesicle-mediated transport Clathrin binding At 5g23690 Polynucleotide RNA processing RNA binding adenylyltransferase protein

Example 15 and other sequence databases using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) Identification of Sequences Related to iSYT (Altschulet al. (1990).J. Mol. Biol. 215:403-410; and Alts 0327 Sequences (full length cDNA, ESTs or genomic) chul et al. (1997) Nucleic Acids Res. 25:3389-3402). The related to iSYT nucleic acid and polypeptides were identified program is used to find regions of local similarity between amongst those maintained in the EntreZNucleotides database sequences by comparing nucleic acid or polypeptide at the National Centerfor Biotechnology Information (NCBI) sequences to sequence databases and by calculating the sta US 2015/0106974 A1 Apr. 16, 2015 tistical significance of matches. For example, the polypeptide 0328 Tables A2 to A26 provides a list of polypeptide encoded by the nucleic acid of SYT was used for the sequences related to the polypeptides of Table A. TBLASTN algorithm, with default settings and the filter to 0329 Sequences have been tentatively assembled and ignore low complexity sequences set off. The output of the publicly disclosed by research institutions, such as The Insti analysis was viewed by pairwise comparison, and ranked tute for Genomic Research (TIGR; beginning with TA). The according to the probability score (E-value), where the score Eukaryotic Gene Orthologs (EGO) database may be used to reflect the probability that a particular alignment occurs by identify such related sequences, either by keyword search or chance (the lower the E-value, the more significant the hit). In by using the BLAST algorithm with the nucleic acid addition to E-values, comparisons were also scored by per sequence or polypeptide sequence of interest. Special nucleic centage identity. Percentage identity refers to the number of acid sequence databases have been created for particular identical nucleotides (or amino acids) between the two com organisms, such as by the Joint Genome Institute. Further pared nucleic acid (or polypeptide) sequences over a particu more, access to proprietary databases, has allowed the iden lar length. In some Instances, the default parameters may be tification of novel nucleic acid and polypeptide sequences. adjusted to modify the stringency of the search. For example the E-value may be increased to show less Stringent matches. Concerning SYT This way, short nearly exact matches may be identified. 0330 TABLE A2 Preferred honologous polypeptides of SYT Name Source organism Database accession number Arath SYT2 Arabidopsis thaliana AY10264.0.1 Arath SYT3 Arabidopsis thaliana AY1O2641.1 Alice SYT2 Allium cepa CF437485 Aqufo SYT1 Aquilegia formosa X Aquilegia pubescens DT758802.1 Aqufo SYT2 Aquilegia formosa X Aquilegia pubescens TA15831 338,618 T25K16.15 Aspof SYT1 Aspergilius officinalis CV287542 BetVu SYT2 Beta vulgaris BQ594749.1 BQ594658.1 Bradi SYT3 Brachypodium distachyon DV48OO64.1 Brana SYT1 Brassica napus CD823592 Brana SYT2 Brassica napa CNf32814 Chlre SYT Chlamydomonas reinhardtii BQ814858, igi Chlre3 194013 estExt fgenesh.2 pg. C 510025 Citsi SYT1 Citrus Sinensis CB290S88 Citsi SYT2 Citrus Sinensis CV7175O1 Cryja SYT1 Cryptomeria japonica TA3001 3369 2 Curlo SYT2 Circlina longa TA2676. 136217 Eupes SYT2 Euphorbia esatia DV144834 Frave SYT2 Fragaria vesca DY668312 Glyma SYT1.1 Glycine max TA55102 3847 Glyma SYT1.2 Glycine max TA51451 3847 Glyma SYT2.1 Glycine max BQ612648 Glyma SYT2.2 Glycine max TA48452 3847 Glyso SYT2 Glycine Sova CA799921 Gosar SYT Gossypium arboretin BM359324 Goshi SYT Gossypium hirsutium DT558852 Goshi SYT2 Gossypium hirsutium DTS63805 Helan SYT Helianthus annuit is TA12738 4232 Horvu SYT2 Hordeum vulgare CAO323SO Lacse SYT2 Lactuca seriola DW110765 Lyces SYT Lycopersicon escientiin AW934450.1 BP89.3155.1 Maldo SYT2 Maius domestica CVO84230 DR997S 66 Medtr SYT Medicago trunctilata CA858SO7.1 Medtr SYT2 Medicago trunctilata CA858743 B310799.1 AL382135.1 Orysa SYT Oryza sativa AKOS8575 Orysa SYT2 Oryza sativa AK105,366 Orysa SYT3 Oryza sativa BP1850O8 Panvi SYT3 Panicum virgatum DN152517 Phypa SYT1.1 Physcomitrella patens TA28566 3218 Phypa SYT1.2 Physcomitrella patens TA21282. 3218 Phypa SYT1.3 Physcomitrella patens TA20922 3218 Phypa SYT1.4 Physcomitrella patens TA29452 3218 Picsi SYT Picea sitchensis DR4841OO DR478464.1 Pinta SYT Pinus taeda DT625916 Poptr SYT Populus trichocarpa DT476906 Poptr SYT2 Populus trichocarpa Scaff XIV493 Poptr SYT1.2 Populus trichocarpa CV257942.1 Prupe SYT2 Prints DT4S4880.1 persica DT4SS286.1 US 2015/0106974 A1 Apr. 16, 2015 34

TABLE A2-continued Preferred honologous polypeptides of SYT Name Source organism Database accession number Sacof SYT1 Saccharum officinarum CAO78249.1 CAO7863O CAO82679 CA234,526 CA239244 CAO83312 Sacof SYT2 Saccharum officinarum CA110367 Sacof SYT3 Saccharum officinarum CA161933.1 CA26SO85 Solitu SYT1.1 Soianum tuberosum CK265597 Solitu SYT1.2 Soianum tuberosum BGS90990 Solitu SYT3 Soianum tuberosum CK272804 Sorbi SYT1 Sorghum bicolor TA40712 4558 Sorbi SYT2 Sorghum bicolor CF482417 CW376.917 Sorbi SYT3 Sorghum bicolor CX611128 Taxof SYT2 Taraxacum officinale TA1299 50225 Taxof SYT3 Taraxacum officinale TASOOO 50225 Triae SYT1 Trictim aestivum TA105893 4565 Triae SYT2 Trictim aestivum CD901951 Triae SYT3 Trictim aestivum BJ246754 BJ25 2709 Vitvi SYT1.1 Vitis vinifera DV219834 Vitvi SYT1.2 Vitis vinifera EE108079 Vitvi SYT2.1 Vitis vinifera EC939550 Vitvi SYT2.2 Vitis vinifera EEO94148.1 EC964169.1 Wolca SYT Voivox carrieri JGI CBHO11121.fwdGI CBHO11121. rew Welmi SYT Weiwitschia mirabilis DTS98761 Zeama SYT1 Zea mayS BG874129.1 CA409022.1 Zeama SYT2 Zea mayS AY106697 Zeama SYT3 Zea mayS CO468901 Homsa SYT Homo sapiens CRS42103

Concerning the AT1G05370 Polypeptide TABLE A3-continued 0331 Preferred honologous polypeptides, of ATIGO8730 polypeptide TABLE A2 SYT Name homologue AT1GO8730.1 >T. aestivum TC3396.58#1 - Preferred homologous polypeptides, of ATIGO5370 polypeptide AT1GO8730.1 >Z. mays TC526723#1 SYT Name homologue AT1GOS370.1 G. max Glyma20g28380.3#1 AT1GOS370.1 M. truncatula AC161569 9.5#1 AT1GOS370.1 O. sativa Os03g0219100th 1 Concerning the AT1G09270 Polypeptide AT1GOS370.1 P. patens NP13131528#1 AT1GOS370.1 P. trichocarpa scaff X.810#1 0333 AT1G05370.1 Z. mays ZMO7MC22382 BFb0062G18(a)22320it 1 TABLE A4 Concerning AT1 G873O Polypeptide Preferred honologous polypeptides, of ATIG09270 polypeptide 0332 SYT Name homologue AT1G09270.1 >B. napus TC77714th 1 TABLE A3 AT1G09270.1 >G. max Glyma.09g.04430.1#1 AT1G09270.1 >G. hirsutum. ES800234#1 Preferred honologous polypeptides, of ATIGO8730 polypeptide AT1G09270.1 >H. vulgare TC178368#1 AT1G09270.1 >M. truncatula AC191599. 15.4f1 SYT Name homologue AT1G09270.1 >O. sativa OsO5g0155500#1 AT1G09270.1 >P. patens TC29288#1 AT1 GO8730.1 >G. max Glyma13g16710.1 #1 AT1G09270.1 >P. trichocarpa scaff XIII.64#1 AT1 GO8730.1 >M. truncatula CUO41231 65.4#1 AT1G09270.1 >S. lycopersicum TC192018#1 AT1 GO8730.1 >O. sativa Os06g0488200+1 AT1G09270.1 >T. aestivum TC326489H1 AT1 GO8730.1 >P. patens TC53472f1 AT1G09270.1 >Z. mays ZMO7MCO9815 62040840(a)9797#1 AT1 GO8730.1 >P. trichocarpa scaff 201.14#1

US 2015/0106974 A1 Apr. 16, 2015 38

Concerning the AT5G23690 Polypeptide TABLE A26-continued 0353 Preferred honologous polypeptides. TABLE A24 SYT Name homologue ATSGSS210 >G. max Glyme18g10110.1 #1 Preferred honologous polypeptides. ATSGSS210 >H. vulgare BG342941 #1 ATSGSS210 >H. vulgare TC167448#1 SYT Name homologue ATSGSS210 >M. truncatula TC122534#1 ATSG-23690 >A. thaliana ATSG23690.1#1 ATSGSS210 >M. truncatula TC127795#1 ATSG-23690 >A. thaliana AT1 G28090.1 #1 ATSGSS210 >O. sativa LOC Os08g25080.1#1 ATSG-23690 >A. thaliana AT3G48830.1#1 ATSGSS210 >O. sativa LOC Os08g25080.2#1 ATSG-23690 >M. truncatula AC151817 35.4#1 ATSGSS210 >O. sativa LOC Os09g11240.1#1 ATSG-23690 >O. Saltiva TC289531#1 ATSGSS210 >P. patens 167013+1 ATSG-23690 >O. Saltiva TC310376#1 ATSGSS210 >P. trichocarpa scaff 166.68#1 ATSG-23690 >P. petens 161847#1 ATSGSS210 >P. trichocarpa scaff I.2546#1 ATSG-23690 >P. trichocarpa scaff XII.984#1 ATSGSS210 >P. trichocarpa scaff XI. 195#1 ATSG-23690 >P. trichocarpa scaff XV.833#1 ATSGSS210 >T. aestivum. CK204604ii1 ATSG-23690 >V. viniferaXM 02266778.1 hypothetical protein ATSGSS210 >T. aestivum TC293410#1 LOC1002591.04 (LOC1002591.04) ATSGSS210 >Z. mays FL309643h1 ATSG-23690 >Z. mays TC467699#1 ATSGSS210 >Z. mays TC479416#1 ATSO23690 >Z. mays TC473496#1

Example 16 Concerning the AT5G53480 Polypeptide 0354) Alignment of an iSYT Polypeptide and Homologues Thereof TABLE A25 0356 Alignment of polypeptide sequences is performed Preferred honologous polypeptides. using the ClustalW 2.0 algorithm of progressive alignment SYT Name homologue (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; ATSGS3480 >AT5G53480.1 Symbols; importin beta-2, putative chr5 Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with 21731242-2173393S FORWARD standard setting (slow alignment, similarity matrix: or Blo ATSGS3480 A. thaliana AT3GO8943.1#1 Sum 62 (if polypeptides are aligned), gap opening penalty 10, ATSGS3480 A. thaliana AT3GO8947.1#1 gap extension penalty: 0.2). Minor manual editing is done to ATSGS3480 >G. max Glyma.04.g41230.1#1 further optimise the alignment. A phylogenetic tree of an ATSGS3480 >G. max Glyma.05.g36630.1#1 ATSGS3480 >G. max Glyma.06g 13620.1#1 iSYT polypeptide and homologues thereof is constructed ATSGS3480 >G. max Glyma.08g02930.1#1 using a neighbour-joining clustering algorithm as provided in ATSGS3480 >H. vulgare cé2767390hv270303(a)6375#1 the AlignX programme from the Vector NTI (Invitrogen). ATSGS3480 >H. vulgare AK249047 ATSGS3480 >O. sativa LOC Os12.g38110.1 #1 ATSGS3480 >P. patens TC30184t 1 Example 17 ATSGS3480 >P. patens TC31822#1 ATSGS3480 >P. trichocarpa scaff VI.900#1 ATSGS3480 >P. trichocarpa scaff XII.230#1 Calculation of Global Percentage Identity Between ATSGS3480 >P. trichocarpa scaff XV.118#1 Polypeptide Sequences ATSGS3480 >P. trichocarpa scaff XVI.1174ii1 ATSGS3480 >S. bicolor XM 002442302.1 0357 Global percentages of similarity and identity between full length polypeptide sequences useful in perform ing the methods of the Invention were determined using one Concerning the AT5G55210 Polypeptide of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 0355 2003 4:29. MatGAT: an application that generates similarity/ identity matrices using protein or DNA sequences. Cam TABLE A26 panella JJ, Bitincka L. Smalley J: software hosted by Ledion Preferred honologous polypeptides. Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre SYT Name homologue alignment of the data. The program performs a series of ATSGSS210 >A. thaliana ATSG55210.1#1 pair-wise alignments using the Myers and Miller global ATSGSS210 >A. thaliana AT4G22320.1#1 alignment algorithm (with a gap opening penalty of 12, and a ATSGSS210 >B. napus EVO25360#1 gap extension penalty of 2), calculates similarity and identity ATSGSS210 >B. napus TC65291#1 ATSGSS210 >B. napus TC67583#1 using for example Blosum 62 (for polypeptides), and then ATSGSS210 >B. napus TC68897#1 places the results in a distance matrix. Sequence similarity is ATSGSS210 >B. napus TC93468th 1 shown in the bottom half of the dividing line and sequence ATSGSS210 >G. max Glyma.08g43440.1#1 identity is shown in the top half of the diagonal dividing line. ATSGSS210 >G. max Glyma.09g.01220.1 #1 ATSGSS210 >G. max Glyma15g12050.1 #1 0358 Parameters used in the comparison are: Scoring matrix: Blosumó2, First Gap: 12, Extending Gap: 2. US 2015/0106974 A1 Apr. 16, 2015 39

Example 18 bases include SWISS-PROT PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large Identification of Domains Comprised in Polypeptide collection of multiple sequence alignments and hidden Sequences Useful in Performing the Methods of the Markov models covering many common protein domains and Invention families. Pfam is hosted at the Sanger Institute server in the 0359. The Integrated Resource of Protein Families, United Kingdom. Interpro is hosted at the European Bioin Domains and Sites (InterPro) database is an integrated inter formatics Institute in the United Kingdom. face for the commonly used signature databases for text- and 0360. The results of the InterPro scan of the polypeptide sequence-based searches. The InterPro database combines sequence representing the iSYT polypeptides of Table A are these databases, which use different methodologies and vary presented in Tables C1 to C20. ing degrees of biological information about well-character 0361. The term iSYT (Interactor of SYT) and “AN3 inter ized proteins to derive protein signatures. Collaborating data actor” as used herein are interchangeable. TABLE C1 InterPro Scan results (maior accession numbers) of the ATIGO5370 polypeptide. AN3 InterPro amino acid start amino acid end interactor Database Domain accession Domain name accession coordinate coordinate AT1GOS370.1 TMHMM tmhmm transmembrane regions NULL 170 191 AT1GOS370.1 TMHMM tmhmm transmembrane regions NULL 144 166 AT1G05370.1 Seg Seg Seg NULL 3.18 330 AT1G05370.1 Seg Seg Seg NULL 235 2S6 AT1G05370.1 Seg Seg Seg NULL 150 163 AT1GOS370.1 HMMPanther PTHR1O174 RETINALDEHYDE BINDING NULL 18 150 PROTEIN-RELATED AT1G05370.1 superfamily SSF46938 CRALTRION-terminal domain IPRO11074 14 83 AT1G05370.1 superfamily SSF52087 CRALTRIO domain IPROO1251 82 149 AT1GO5370.1 ProfileScan PS5O191 CRAL TRIO IPROO1251 8O 262 AT1GOS370.1 HMMSmart SMOOS16 no description IPROO1251 86 229 AT1GO53701 Gene3D G3DSA:3.40.525.10 description IPROO1251 70 149 O AT1GO8730.1 superfamily SSFS4849 GroEL-intermediate domain like NULL 940 1017

TABLE C2 InterPro Scan results (major accession numbers) of the ATIGO8730 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT1 GO8730. Superfamily SSFS4849 GroEL-intermediate domain like NULL 940 1017 AT1 GO8730. Superfamily SSF52540 P-loop containing nucleoside NULL 798 893 triphosphate hydrolases AT1 GO8730. Superfamily SSF52540 P-loop containing nucleoside NULL 38 797 triphosphate hydrolases AT1 GO8730. SignalPHMM signalp signal-peptide NULL 1 23 AT1 GO8730. Seg Seg Seg NULL 1.191 1208 AT1 GO8730. Seg Seg Seg NULL 101S 1037 AT1 GO8730. Seg Seg Seg NULL 950 966 AT1 GO8730. Seg Seg Seg NULL 60S 618 AT1 GO8730. HMMPanther PTHR13140:SF36 MYOSINXI NULL 1130 1517 AT1 GO8730. HMMPanther PTHR13140:SF36 MYOSINXI NULL 870 925 AT1 GO8730. HMMPanther PTHR13140:SF36 MYOSINXI NULL 312 840 AT1 GO8730. HMMPanther PTHR13140:SF36 MYOSINXI NULL 17 292 AT1 GO8730. HMMPanther PTHR13140 MYOSIN NULL 1130 1517 AT1 GO8730. HMMPanther PTHR13140 MYOSIN NULL 870 925 AT1 GO8730. HMMPanther PTHR13140 MYOSIN NULL 312 840 AT1 GO8730. HMMPanther PTHR13140 MYOSIN NULL 17 292 AT1 GO8730. Gene3D G3DSA:4.10.270.2Ono description NULL 726 771 AT1 GO8730. Gene3D G3DSA:3.30.538.1 Ono description NULL 71 3O4 AT1 GO8730. Gene3D G3DSA:3.30.1370.40no description NULL 655 721 AT1 GO8730. Gene3D G3DSA:110.4651Ono description NULL 446 628 AT1 GO8730. Gene3D G3DSA:1.10.1831Ono description NULL 32O 428 AT1 GO8730. Superfamily SSFSOO84 Myosin S1 fragment, N-terminal domain IPRO08989 13 37 AT1 GO8730. HMMPfan PFO2736 Myosin N IPROO4009 18 59 AT1 GO8730. ProfileScan PS51126 DILUTE IPROO2710 1168 1481 AT1 GO8730. HMMPfan PFO1843 DIL IPROO2710 1356 1463 AT1 GO8730. BlastProDon PDOO3376 Q6ZGC5 EEEEE Q6ZGC5; IPROO2710 1287 1434 AT1 GO8730. HMMSmart SMOO242 no description IPROO1609 64 741 AT1 GO8730. HMMPfan PFOOO63 Myosin head IPROO1609 72 728 AT1 GO8730. FPrintScan PROO193 MYOSINHEAVY IPROO1609 490 518 US 2015/0106974 A1 Apr. 16, 2015 40

TABLE C2-continued InterPro scan results (major accession numbers) of the ATIGO8730 polypeptide. AN3 interPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT1GO8730. FPrintScan PROO193 MYOSINHEAVY PROO1609 437 465 AT1GO8730. FPrintScan PROO193 MYOSINHEAVY PROO1609 204 231 AT1GO8730. FPrintScan PROO193 MYOSINHEAVY PROO1609 157 182 AT1GO8730. FPrintScan PROO193 MYOSINHEAVY PROO1609 100 119 AT1GO8730. BlastProDOn PDOOO3SS Q97.VN3 ARATH Q97.VN3; PROO1609 196 226 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 862 891 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 839 866 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 814 843 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 791 818 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 766 795 AT1GO8730. ProfileScan PS50096 IQ PROOOO48 743 772 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 861 883 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 838 860 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 813 835 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 790 812 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 765 787 AT1GO8730. HMMSmart SMOOO15 no description PROOOO48 742 764 AT1GO8730. HMMPfan PFOO612 IQ PROOOO48 840 860 AT1GO8730. HMMPfan PFOO612 IQ PROOOO48 815 835 AT1GO8730. HMMPfan PFOO612 IQ PROOOO48 792 812 AT1GO8730. HMMPfan PFOO612 IQ PROOOO48 767 787 AT1GO8730. HMMPfan PFOO612 IQ PROOOO48 744 764

TABLE C3 InterPro scan results (major accession numbers) of the ATIG09270 polypeptide. AN3 interPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT1GO927O. HMMPanther PTHR23316 PTHR23316 NULL 1 537 AT1GO927O. HMMPanther PTHR23316 PTHR23316 NULL 1 537 AT1GO927O. Superfamily SSF48371 ARM-type fold IPRO16024 49 5O1 AT1GO927O. Gene3D G3DSA:1.25.10.10ARM-like PRO11989 81 5O1 AT1GO927O. ProfileScan PSS1214 IBB PROO2652 1 58 AT1GO927O. HMMPfan PFO1749 IBB PROO2652 4 102 AT1GO927O. Gene3D G3DSA:1.20.5.690Importin-a-like IBB- PROO2652 9 52 bd AT1GO927O. ProfileScan PS5O176 ARM REPEAT IPROOO225 333 376 AT1GO927O. ProfileScan PS5O176 ARM REPEAT IPROOO225 16S 2O7 AT1GO927O. ProfileScan PS5O176 ARM REPEAT IPROOO225 122 16S AT1GO927O. HMMSmart SMOO185 ARM PROOO225 408 448 AT1GO927O. HMMSmart SMOO185 ARM PROOO225 365 40S AT1GO927O. HMMSmart SMOO185 ARM PROOO225 322 363 AT1GO927O. HMMSmart SMOO185 ARM PROOO225 28O 32O AT1GO927O. HMMSmart SMOO185 ARM PROOO225 239 278 AT1GO927O. HMMSmart SMOO185 ARM PROOO225 195 237 AT1GO927O. HMMSmart SMOO185 ARM PROOO225 154 194 AT1GO927O. HMMSmart SMOO185 ARM PROOO225 111 152 AT1GO927O. HMMPfan PF00514 Arm PROOO225 408 448 AT1GO927O. HMMPfan PF00514 Arm PROOO225 365 40S AT1GO927O. HMMPfan PF00514 Arm PROOO225 322 363 AT1GO927O. HMMPfan PF00514 Arm PROOO225 28O 32O AT1GO927O. HMMPfan PF00514 Arm PROOO225 239 278 AT1GO927O. HMMPfan PF00514 Arm PROOO225 196 237 AT1GO927O. HMMPfan PF00514 Arm PROOO225 154 194 AT1GO927O. HMMPfan PF00514 Arm PROOO225 111 152

TABLE C4

InterPro scan results (maior accession numbers) of the AT1 G18450. polypeptide.

AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT1 G1845O1 Superfamily SSF53067 SSF53067 NULL 170 441 AT1 G1845O1 HMMPanther PTHR11937:SF32 PTHR11937:SF32 NULL 254 441 US 2015/0106974 A1 Apr. 16, 2015 41

TABLE C4-continued

InterPro scan results (maior accession numbers) of the AT1 G18450. polypeptide.

AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT1 G1845O1 Superfamily SSF53067 SSF53067 NULL 3 170 AT1 G1845O1 HMMPanther PTHR11937:SF32 PTHR11937:SF32 NULL 74 223 AT1 G1845O1 HMMPanther PTHR11937:SF32 PTHR11937:SF32 NULL 31 441 AT1 G1845O1 HMMPanther PTHR11937:SF32 PTHR11937:SF32 NULL 31 52 AT1 G1845O1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 404 441 AT1 G1845O1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 296 387 AT1 G1845O1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 3 164 AT1 G1845O1 HMMSmart SMOO268 ACTIN IPROO4OOO 7 441 AT1 G1845O1 HMMPfan PFOOO22 Actin IPROO4OOO 4 441 AT1 G1845O1 HMMPanther PTHR11937 Actin like IPROO4OOO 31 441

TABLE C5 InterPro scan results (major accession numbers) of the ATIG20670 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT1G2O670.1 HMMPanther PTHR22881:SF3 PTHR22881:SF3 NULL 173 652 AT1G2O670.1 HMMPanther PTHR22881:SF3 PTHR22881:SF3 NULL 173 652 AT1G2O670.1 HMMPanther PTHR22881 PTHR22881 NULL 173 652 AT1G2O670.1 HMMPanther PTHR22881 PTHR22881 NULL 173 652 AT1G2O670.1 Superfamily SSF47370 Bromodomain IPROO1487 145 288 AT1G2O670.1 ProfileScan PSSOO14 BROMODOMAIN 2 IPROO1487 188 258 AT1G2O670.1 ProfileScan PSOO633 BROMODOMAIN 1 IPROO1487 194 250 AT1G2O670.1 HMMSmart SMOO297 BROMO IPROO1487 169 277 AT1G2O670.1 HMMPfan PFOO439 Bromodomain IPROO1487 176 263 AT1G2O670.1 Gene3D G3DSA:1.20.920.10Bromodomain IPROO1487 152 306 AT1G2O670.1 FPrintScan PROO503 BROMODOMAIN IPROO1487 239 258 AT1G2O670.1 FPrintScan PROO503 BROMODOMAIN IPROO1487 221 239 AT1G2O670.1 FPrintScan PROO503 BROMODOMAIN IPROO1487 205 221

TABLE C6 InterPro Scan results (maior accession numbers) of the ATIG21700 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT1G21700.1 HMMPanther PTHR128O2:SF4 PTHR12802:SF4 NULL 175 807 AT1G21700.1 HMMPanther PTHR128O2:SF4 PTHR12802:SF4 NULL 175 807 AT1G21700.1 HMMPanther PTHR128O2 PTHR12802 NULL 175 807 AT1G21700.1 HMMPanther PTHR128O2 PTHR12802 NULL 175 807 AT1G21700.1 HMMPfan PFOO249 Myb DNA-binding IPRO14778 400 445 AT1G21700.1 Superfamily SSF46689 Homeodomain like IPROO9057 394 447 AT1G21700.1 ProfileScan PSSO934 SWIRM IPROO7526 176 274 AT1G21700.1 HMMPfan PFO4433 SWIRM IPROO7526 176 26S AT1G21700.1 ProfileScan PS50090 MYB 3 IPROO1OOS 4O2 445 AT1G21700.1 HMMSmart SMOO717 SANT IPROO1OOS 399 447

TABLE C7 InterPro Scan results (major accession numbers) of the ATIG23900 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT1G-239002 HMMPanther PTHR22780:SFS PTHR22780:SF5 NULL 10 876 AT1G-239002 HMMPanther PTHR22780:SFS PTHR22780:SF5 NULL 10 876 AT1G-239002 HMMPanther PTHR22780 PTHR22780 NULL 10 876 AT1G-239002 HMMPanther PTHR22780 PTHR22780 NULL 10 876 AT1G-239002 HMMPIR PIRSFO37094 AP1 complex gamma IPRO17107 1 876 AT1G23900.2 Superfamily SSF48371 ARM-type fold IPRO16024 6 591 AT1G23900.2 Superfamily SSF49348 Clath adapt IPRO 13041 726 873 AT1G-239002 Gene3D G3DSA:1.25.10.10ARM-like IPRO11989 9 572 US 2015/0106974 A1 Apr. 16, 2015 42

TABLE C7-continued InterPro Scan results (major accession numbers) of the ATIG23900 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT1G23900.2 ProfileScan PS5018O GAE IPROO8153 756 873 AT1G-239002 Gene3D G3DSA:26O4(O.1230 IPROO8153 757 873 Clathrin g-adaptin app AT1G-239002 BlastProDOn PDO2.1457 Gamma adaptin C IPROO8153 760 873 AT1G-239002 HMMSmart SMOO809 Alpha adaptinc2 IPROO8152 753 873 AT1G-239002 HMMPfan PFO2883 Alpha adaptinc2 IPROO8152 753 873 AT1G-239002 HMMPfan PFO16O2 Adaptin N IPROO2553 26 S8O

TABLE C8 InterPro Scan results (major accession numbers) of the ATIG65980 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT1G6598O2. HMMPanther PTHR10430:SF4 PTHR10430:SF4 NULL 1 104 AT1G6598O2. HMMPanther PTHR10430:SF4 PTHR10430:SF4 NULL 1 104 AT1G6598O2. HMMPanther PTHR10430 PTHR10430 NULL 1 104 AT1G6598O2. HMMPanther PTHR10430 PTHR10430 NULL 1 104 AT1G6598O2. HMMPfan PFO8534 Redoxin IPRO 13740 5 120 AT1G65980.2 superfamily SSF52833 Thiordxn-like fil IPRO12336 4 103 AT1G6598O.2 Gene3D G3DSA:3.40.30.1OThioredoxin fold IPRO12335 2 104

TABLE C9 InterPro Scan results (maior accession numbers) of the AT2G18876 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT2G18876.2 HMMPanther PTHR21736:SF12 PTHR21736:SF12 NULL 64 284 AT2G18876.2 HMMPanther PTHR21736:SF12 PTHR21736:SF12 NULL 64 284 AT2G18876.2 HMMPanther PTHR21736 PTHR21736 NULL 64 284 AT2G18876.2 HMMPanther PTHR21736 PTHR21736 NULL 64 284

TABLE C10 InterPro Scan results (maior accession numbers) of the AT2G4.6020 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT2G46O2O2 Superfamily SSF52540 SSF52540 NULL 1300 434 AT2G46O2O2 Superfamily SSF52540 SSF52540 NULL 758 157 AT2G46O2O2 HMMPanther PTHR10799:SF76 PTHR10799:SF76 NULL 146S 721 AT2G46O2O2 HMMPanther PTHR10799:SF76 PTHR10799:SF76 NULL 96S 430 AT2G46O2O2 HMMPanther PTHR10799:SF76 PTHR10799:SF76 NULL 2O2 217 AT2G46O2O2 HMMPanther PTHR10799:SF76 PTHR10799:SF76 NULL 24 721 AT2G46O2O2 HMMPanther PTHR10799:SF76 PTHR10799:SF76 NULL 24 74 AT2G46O2O2 HMMPanther PTHR10799 PTHR1O799 NULL 146S 721 AT2G46O2O2 HMMPanther PTHR10799 PTHR1O799 NULL 96S 430 AT2G46O2O2 HMMPanther PTHR10799 PTHR1O799 NULL 2O2 217 AT2G46O2O2 HMMPanther PTHR10799 PTHR1O799 NULL 24 721 AT2G46O2O2 HMMPanther PTHR10799 PTHR1O799 NULL 24 74 AT2G46O2O2 Gene3D G3DSA:34O.S.O.3OOG3DSA:3.4O.S.O.300 NULL 1309 444 AT2G46O2O2 HMMPfan PFO888O QLQ IPRO14978 462 499 AT2G46O2O2 ProfileScan PS51.192 HELICASE ATP IPRO14021 993 158 BIND 1 AT2G46O2O2 HMMSmart SMOO487 DEXDC IPRO14001 977 166 AT2G46O2O2 ProfileScan PSS1194 HELICASE CTER IPROO1650 1312 489 AT2G46O2O2 HMMSmart SMOO490 HELICC IPROO16SO 1338 422 AT2G46O2O2 HMMPfan PFOO271 Helicase C IPROO16SO 1343 422 AT2G46O2O2 Superfamily SSF47370 Bromodomain IPROO1487 1890 2010 AT2G46O2O2 HMMSmart SMOO297 BROMO IPROO1487 1900 2007 US 2015/0106974 A1 Apr. 16, 2015 43

TABLE C10-continued InterPro scan results (major accession numbers) of the AT2G4.6020 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT2G46O2O2 Gene3D G3DSA:1.20.920.10Bromodomain IPROO1487 1889 2003 AT2G46O2O2 HMMPfan PFOO176 SNF2 N IPROOO330 984 1291

TABLE C11 InterPro scan results (major accession numbers) of the AT3GO6720 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT3GO6720. HMMPanther PTHR23316 PTHR23316 NULL 1 531 AT3GO6720. HMMPanther PTHR23316 PTHR23316 NULL 1 531 AT3GO6720. Superfamily SSF48371 ARM-type fold PRO16024 43 494 AT3GO6720. Gene3D G3DSA:1.25.10.1OARM- PRO11989 72 494 like AT3GO6720. ProfileScan PSS1214 IBB PROO2652 1 58 AT3GO6720. HMMPfan PFO1749 IBB PROO2652 4 95 AT3GO6720. ProfileScan PS5O176 ARM REPEAT PROOO225 326 36 AT3GO6720. ProfileScan PS5O176 ARM REPEAT PROOO225 242 284 AT3GO6720. ProfileScan PS5O176 ARM REPEAT PROOO225 158 186 AT3GO6720. ProfileScan PS5O176 ARM REPEAT PROOO225 115 154 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 401 44 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 358 398 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 315 356 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 273 313 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 232 27 AT3GO672O. HMMSmart SMOO185 ARM PROOO225 188 230 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 147 187 AT3GO6720. HMMSmart SMOO185 ARM PROOO225 104 145 AT3GO6720. HMMPfan PF00514 Arm PROOO225 401 44 AT3GO6720. HMMPfan PF00514 Arm PROOO225 358 398 AT3GO6720. HMMPfan PF00514 Arm PROOO225 315 356 AT3GO6720. HMMPfan PF00514 Arm PROOO225 273 313 AT3GO6720. HMMPfan PF00514 Arm PROOO225 232 27 AT3GO6720. HMMPfan PF00514 Arm PROOO225 189 230 AT3GO6720. HMMPfan PF00514 Arm PROOO225 147 187 AT3GO6720. HMMPfan PF00514 Arm PROOO225 104 145

TABLE C12 InterPro Scan results (major accession numbers) of the AT3G15000 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT3G 15000.1 Seg Seg Seg NULL 311 385 AT3G 15000.1 Seg Seg Seg NULL 28O 3O2 AT3G 15000.1 Seg Seg Seg NULL 239 278 AT3G 15000.1 Seg Seg Seg NULL 2O6 229 AT3G 15000.1 Seg Seg Seg NULL 25 50

TABLE C13 InterPro scan results (major accession numbers) of the AT3G60830 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT3G60830.1 Superfamily SSF53067 SSF53067 NULL 135 363 AT3G60830.1 Superfamily SSF53067 SSF53067 NULL 1 141 AT3G60830.1 HMMPanther PTHR11937:SF26 PTHR11937:SF26 NULL 2O 363 AT3G60830.1 HMMPanther PTHR11937:SF26 PTHR11937:SF26 NULL 2O 363 AT3G60830.1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 325 363 AT3G60830.1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 228 3OO AT3G60830.1 Gene3D G3DSA:3.30.420.4OG3DSA:33O420.40 NULL 3 128 US 2015/0106974 A1 Apr. 16, 2015 44

TABLE C13-continued

InterPro scan results (major accession numbers) of the AT3G60830 polypeptide.

AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

AT3G60830.1 HMMSmart SMOO268 ACTIN IPROO4OOO 1 363 AT3G60830.1 HMMPfan PFOOO22 Actin IPROO4OOO 3 363 AT3G60830.1 HMMPanther PTHR11937 Actin like IPROO4OOO 2O 363 AT3G60830.1 FPrintScan PROO190 ACTIN IPROO4OOO 219 235 AT3G60830.1 FPrintScan PROO190 ACTIN IPROO4OOO 128 147 AT3G60830.1 FPrintScan PROO190 ACTIN IPROO4OOO 103 116 AT3G60830.1 FPrintScan PROO190 ACTIN IPROO4OOO 23 32

TABLE C14 InterPro Scan results (maior accession numbers) of the AT4G16143 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT4G16143. Seg Seg Seg NULL 333 340 AT4G16143. HMMPanther PTHR23316 IMPORTIN NULL 1 534 ALPHA AT4G16143. Superfamily SSF48371 ARM repeat PRO16024 77 499 AT4G16143. Gene3D G3DSA:1.25.10.1Ono description PRO11989 77 499 AT4G16143. ProfileScan PSS1214 IBB PROO2652 1 58 AT4G16143. HMMPfan PFO1749 IBB PROO2652 4 1OO AT4G16143. ProfileScan PS5O176 ARM REPEAT IPROOO225 331 374 AT4G16143. ProfileScan PS5O176 ARM REPEAT IPROOO225 289 331 AT4G16143. ProfileScan PS5O176 ARM REPEAT IPROOO225 247 289 AT4G16143. ProfileScan PS5O176 ARM REPEAT IPROOO225 163 205 AT4G16143. ProfileScan PS5O176 ARM REPEAT IPROOO225 120 163 AT4G16143. HMMSmart SMOO185 no description PROOO225 406 446 AT4G16143. HMMSmart SMOO185 no description PROOO225 363 403 AT4G16143. HMMSmart SMOO185 no description PROOO225 320 361 AT4G16143. HMMSmart SMOO185 no description PROOO225 278 318 AT4G16143. HMMSmart SMOO185 no description PROOO225 237 276 AT4G16143. HMMSmart SMOO185 no description PROOO225 193 235 AT4G16143. HMMSmart SMOO185 no description PROOO225 152 192 AT4G16143. HMMSmart SMOO185 no description PROOO225 109 150 AT4G16143. HMMPfan PF00514 Arm PROOO225 406 446 AT4G16143. HMMPfan PF00514 Arm PROOO225 363 403 AT4G16143. HMMPfan PF00514 Arm PROOO225 320 361 AT4G16143. HMMPfan PF00514 Arm PROOO225 278 318 AT4G16143. HMMPfan PF00514 Arm PROOO225 237 276 AT4G16143. HMMPfan PF00514 Arm PROOO225 194 235 AT4G16143. HMMPfan PF00514 Arm PROOO225 152 192 AT4G16143. HMMPfan PF00514 Arm PROOO225 109 150

TABLE C15 InterPro scan results (major accession numbers) of the AT4G21540 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT4G21540.1 SignalPHMM signalp signal-peptide NULL 1 30 AT4G21540.1 Seg Seg Seg NULL 226 236 AT4G21540.1 Seg Seg Seg NULL 19 37 AT4G21540.1 HMMPanther PTHR12358:SF1 O SPHINGOSINEKINASE- NULL 373 747 RELATED AT4G21540.1 HMMPanther PTHR12358 SPHINGOSINEKINASE NULL 373 747 AT4G21540.1 HMMSmart SMOOO46 no description IPROO12O6 909 1041 AT4G21540.1 HMMSmart SMOOO46 no description IPROO12O6 379 515 AT4G21540.1 HMMPfam PFOO781 DAGK cat IPROO12O6 909 1041 AT4G21540.1 HMMPfam PFOO781 DAGK cat IPROO12O6 379 515 AT4G21540.1 BlastProDom PD005043 O65419 ARATH O65419; IPROO1206 908 1018 AT4G21540.1 BlastProDom PD005043 O65419 ARATH O65419; IPROO1206 378 488 US 2015/0106974 A1 Apr. 16, 2015 45

TABLE C16

InterPro scan results (major accession numbers) of the AT4G27550 polypeptide.

AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

Superfamily SSFS6784 SSFS6784 NULL 511 790 Superfamily SSF53756 SSF53756 NULL 4 467 HMMPanther PTHR1O788 PTHR1O788 NULL 68 730 HMMPanther PTHR1O788 PTHR1O788 NULL 68 730 Gene3D G3DSA:34O.S.O.2OOOG3DSA:3.40.50.2OOO NULL 247 452 HMMTigr TIGROO685 T6PP IPROO3337 SO8 791 HMMPfan PFO2358 Trehalose PPase IPROO3337 S14 758 HMMPfan PFOO982 Glyco transf 20 IPROO1830 469

TABLE C17 InterPro scan results (major accession numbers) of the ATSG13030 polypeptide.

AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

ATSG13O30.1 HMMPfan PFO2696 UPFOO61 IPROO3846 108 604 ATSG1417.0.1 HMMPanther PTHR13844 PTHR13844 NULL 250 523 ATSG1417.0.1 HMMPanther PTHR13844 PTHR13844 NULL 250 523 ATSG1417.0.1 Superfamily SSF47592 MDM2 IPROO3121 3O8 400 ATSG1417.0.1 HMMPfan PFO22O1 SWIB IPROO3121 315 390 ATSG1417.0.1 Gene3D G3DSA:1.10.245.1OSWIB MDM2 IPROO3121 3O8 400

TABLE C18 InterPro Scan results (major accession numbers) of the ATSG 17510 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate AT5G17510.1 Seg Seg Seg NULL 343 357 AT5G17510.1 Seg Seg Seg NULL 70 121 AT5G17510.1 Seg Seg Seg NULL 15 51 AT5G17510.1 superfamily SSF47175 Cytochromes IPRO10980 12 151

TABLE C19 InterPro scan results (major accession numbers) of the ATSG23690 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate ATSG-23690.1 Superfamily SSF81891 Poly A polymerase C-terminal NULL 224 443 region-like ATSG-23690.1 Superfamily SSF81301 Nucleotidyltransferase NULL 76 223 ATSG-23690.1 Seg Seg Seg NULL 240 252 ATSG-23690.1 HMMPanther PTHR13734:SF12 POLY(A) POLYMERASE, NULL 112 523 ARABIDOPSIS ATSG-23690.1 HMMPanther PTHR13734 TRNA-NUCLEO- NULL 112 523 TIDYLTRANSFERASE/POLY(A) POLYMERASE FAMILYMEMBER ATSG-23690.1 Gene3D G3DSA:3.30.460.1Ono description NULL 66 223 ATSG-23690.1 Gene3D G3DSA:110.30901 Ono description NULL 224 316 ATSG-23690.1 ScanRegExp PSOOO12 PHOSPHOPANTETHEINE IPROO6162 71 86 ATSG-23690.1 HMMPfan PFO1743 PolyA pol IPROO2646 97 226 ATSG-23690.1 ScanRegExp PSOOO14 ER TARGET IPROOO886 524 527 US 2015/0106974 A1 Apr. 16, 2015 46

TABLE C2O InterPro Scan results (major accession numbers) of the ATSG53480 polypeptide. AN3 InterPro amino acid amino acid interactor Database Domain accession Domain name accession start coordinate end coordinate

ATSGS3480.1 HMMPanther PTHR1OS27:SF1 PTHR10527:SF1 NULL 217 869 ATSGS3480.1 HMMPanther PTHR1OS27:SF1 PTHR10527:SF1 NULL 217 869 ATSGS3480.1 HMMPanther PTHR1OS27 PTHR10527 NULL 217 869 ATSGS3480.1 HMMPanther PTHR1OS27 PTHR10527 NULL 217 869 AT5G53480.1 superfamily SSF48371 ARM-type fold IPRO16024 3 864 ATSGS3480.1 Gene3D G3DSA:1.25.10.1OARM- IPRO11989 3 866 like ATSG53480.1 ProfileScan PSSO166 IMPORTIN B NT IPROO1494 23 103 ATSGS3480.1 HMMPfan PFO3810 IBN N IPROO1494 23 103 ATSGS3480.1 HMMPfan PFO2985 HEAT IPROOO357 404 441 ATSGS3480.1 HMMPfan PFO2985 HEAT IPROOO357 362 398 ATSGS3480.1 HMMPfan PFO2985 HEAT IPROOO357 214 250

Example 19 0373) Each plant transformation vector was constructed in two steps: First, the two coding sequences of gene of interest Topology Prediction of the iSYT Polypeptide were amplified from the cDNA obtained from the appropriate Sequences source using a high-fidelity PCR. To this end primers were 0362 TargetP 1.1 predicts the subcellular location of designed and synthesized using standard methods. Then the eukaryotic proteins. The location assignment is based on the sequences were cloned in poONR201 P1-P4 and predicted presence of any of the N-terminal pre-sequences: pDONR201 P3-P2 (InvitrogenTM), respectively, using the chloroplast transit peptide (cTP), mitochondrial targeting GatewayTM BP (InvitrogenTM) standard reaction method. The peptide (mTP) or secretory pathway signal peptide (SP). resulting clone was called the Entry Clone (EC), in accor Scores on which the final prediction is based are not really dance with Gateway method terminology. The other entry probabilities, and they do not necessarily add to one. How clone that carried a terminator and a promoter was produced ever, the location with the highest score is the most likely by using plDONR201 P4r-P3r. The identity of the clone was according to TargetP and the relationship between the scores Verified by restriction digestion analysis and complete (the reliability class) may be an indication of how certain the sequencing of the insert. After verification, the clone (EC) prediction is. The reliability class (RC) ranges from 1 to 5, went through a second Gateway method step which allows where 1 indicates the strongest prediction. TargetP is main transfer of the inserts of all 3 ECs to the so-called Destination tained at the server of the Technical University of Denmark. Vector (DV) using the Gateway LR (InvitrogenTM) standard 0363 For the sequences predicted to contain an N-termi reaction method. On the destination vector, a promoter and a nal presequence a potential cleavage site can also be pre terminator designed for stacking genes were already in place. dicted. The identity of the resulting clone was verified by restriction 0364. A number of parameters is selected. Such as organ digestion analysis and then by sequencing. After this verifi ism group (non-plant or plant), cutoffsets (none, predefined cation, this binary vector was used as plant transformation set of cutoffs, or user-specified set of cutoffs), and the calcu vector. The plant transformation vector contains following lation of prediction of cleavage sites (yes or no). functional cassettes in its t-DNA region: the selectable 0365. Many other algorithms can be used to perform such marker gene, the visual (reporter) marker gene, and two genes analyses, including: of interest. Each of these genes was driven by its correspond 0366 ChloroP 1.1 hosted on the server of the Technical ing promoter and terminator. The binary vector is then cloned University of Denmark; into a disarmed Agrobacterium tumefaciens which Is used to 0367 Protein Prowler Subcellular Localisation Predic transform rice. tor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Bris Example 21 bane, Australia; 0368 PENCE Proteome Analyst PA-GOSUB 2.5 Plant Transformation hosted on the server of the University of Alberta, Edm onton, Alberta, Canada; Rice Transformation 0369 TMHMM, hosted on the server of the Technical University of Denmark 0374. The Agrobacterium containing the expression vec 0370 PSORT (URL: psort.org) tor is used to transform Oryza sativa plants. Mature dry seeds 0371 PLOC (Park and Kanehisa, Bloinformatics, 19, of the rice Japonica cultivar Nipponbare are dehusked. Ster 1656-1663, 2003). ilization is carried out by Incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl, followed by Example 20 a 6 times 15 minutes wash with sterile distilled water. The sterile seeds are then germinated on a medium containing Cloning of the iSYT Encoding Nucleic Acid 2,4-D (callus Induction medium). After incubation in the dark Sequence for four weeks, embryogenic, scutellum-derived calli are 0372. The method was adapted from the Multisite Gate excised and propagated on the same medium. After two way.R. Pro (InvitrogenTM). weeks, the calli are multiplied or propagated by Subculture on US 2015/0106974 A1 Apr. 16, 2015 47 the same medium for another 2 weeks. Embryogenic callus Wheat Transformation pieces are sub-cultured on fresh medium 3 days before co 0378 Transformation of wheat is performed with the cultivation (to boost cell division activity). method described by Ishida et al. (1996) Nature Biotech 0375 Agrobacterium strain LBA4404 containing the 14(6): 745-50. The cultivar Bobwhite (available from CIM expression vector is used for co-cultivation. Agrobacterium MYT, Mexico) is commonly used in transformation. Imma was inoculated on AB medium with the appropriate antibiot ture embryos are co-cultivated with Agrobacterium tumefa ics and cultured for 3 days at 28°C. The bacteria are then ciens containing the expression vector, and transgenic plants collected and Suspended in liquid co-cultivation medium to a are recovered through organogenesis. After incubation with density (ODoo) of about 1. The Suspension is then transferred Agrobacterium, the embryos are grown in vitro on callus to a Petridish and the calli immersed in the suspension for 15 induction medium, then regeneration medium, containing the minutes. The callus tissues are then blotted dry on a filter selection agent (for example imidazolinone but various selec paper and transferred to solidified, co-cultivation medium tion markers can be used). The Petri plates are incubated in and incubated for 3 days in the dark at 25°C. Co-cultivated the light at 25°C. for 2-3 weeks, or until shoots develop. The calli are grown on 2,4-D-containing medium for 4 weeks in green shoots are transferred from each embryo to rooting the dark at 28°C. in the presence of a selection agent. During medium and incubated at 25° C. for 2-3 weeks, until roots this period, rapidly growing resistant callus islands devel develop. The rooted shoots are transplanted to soil in the oped. After transfer of this material to a regeneration medium greenhouse. T1 seeds are produced from plants that exhibit and incubation in the light, the embryogenic potential is tolerance to the selection agent and that contain a single copy released and shoots developed in the next four to five weeks. of the T-DNA insert. Shoots are excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they are Soybean Transformation transferred to soil. Hardened shoots era grown under high humidity and short days in a greenhouse. 0379 Soybean is transformed according to a modification 0376 Approximately 35 independent TO rice transfor of the method described in the Texas A&M patent U.S. Pat. mants are generated for one construct. The primary transfor No. 5,164.310. Several commercial soybean varieties are mants are transferred from a tissue culture chamber to a amenable to transformation by this method. The cultivar Jack greenhouse. After a quantitative PCR analysis to Verify copy (available from the Illinois Seed foundation) is commonly number of the T-DNA insert, only single copy transgenic used for transformation. Soybean seeds are sterilised for in plants that exhibit tolerance to the selection agent are kept for vitro sowing. The hypocotyl, the radicle and one cotyledon harvest of T1 seed. Seeds are then harvested three to five are excised from seven-day old young seedlings. The epicotyl months after transplanting. The method yielded single locus and the remaining cotyledon are further grown to develop transformants at a rate of over 50% (Aldemita and Hodges axillary nodes. These axillary nodes are excised and incu 1996, Chanet al. 1993, Hiei et al. 1994). bated with Agrobacterium tumefaciens containing the expres sion vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated Example 22 shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium Transformation of Other Crops until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that Corn Transformation exhibit tolerance to the selection agent and that contain a 0377 Transformation of maize (Zea mays) is performed single copy of the T-DNA insert. with a modification of the method described by Ishida et al. Rapeseed/Canola Transformation (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are 0380 Cotyledonary petioles and hypocotyls of 5-6 day old amenable to transformation and regeneration. The inbred line young seedling are used as explants for tissue culture and A188 (University of Minnesota) or hybrids with A188 as a transformed according to Babic et al. (1998, Plant Cell Rep parent are good sources of donor material for transformation, 17: 183-188). The commercial cultivar Westar (Agriculture but other genotypes can be used Successfully as well. Ears are Canada) is the standard variety used for transformation, but harvested from corn plant approximately 11 days after polli other varieties can also be used. Canola seeds are surface nation (DAP) when the length of the immature embryo is sterilized for in vitro sowing. The cotyledon petiole explants about 1 to 1.2 mm. Immature embryos are cocultivated with with the cotyledon attached are excised from the in vitro Agrobacterium tumefaciens containing the expression vector, seedlings, and inoculated with Agrobacterium (containing and transgenic plants are recovered through organogenesis. the expression vector) by dipping the cut end of the petiole Excised embryos are grown on callus induction medium, then explant into the bacterial Suspension. The explants are then maize regeneration medium, containing the selection agent cultured for 2 days on MSBAP-3 medium containing 3 mg/1 (for example imidazolinone but various selection markers can BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After be used). The Petri plates are incubated in the light at 25°C. two days of co-cultivation with Agrobacterium, the petiole for 2-3 weeks, or until shoots develop. The green shoots are explants are transferred to MSBAP-3 medium containing 3 transferred from each embryo to maize rooting medium and mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) incubated at 25°C. for 2-3 weeks, until roots develop. The for 7 days, and then cultured on MSBAP-3 medium with rooted shoots are transplanted to soil in the greenhouse. T1 cefotaxime, carbenicillin, or timentin and selection agent seeds are produced from plants that exhibit tolerance to the until shoot regeneration. When the shoots are 5-10 mm in selection agent and that contain a single copy of the T-DNA length, they are cut and transferred to shoot elongation insert. medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of US 2015/0106974 A1 Apr. 16, 2015 48 about 2 cm in length are transferred to the rooting medium length are transferred to tubes with SH medium in fine ver (MSO) for root induction. The rooted shoots are transplanted miculite, Supplemented with 0.1 mg/l indole acetic acid. 6 to soil in the greenhouse. T1 seeds are produced from plants furfurylaminopurine and gibberellic acid. The embryos are that exhibit tolerance to the selection agent and that contain a cultivated at 30°C. with a photoperiod of 16 hrs, and plantlets single copy of the T-DNA insert. at the 2 to 3 leaf stage are transferred to pots with vermiculite and nutrients. The plants are hardened and Subsequently Alfalfa Transformation moved to the greenhouse for further cultivation. 0381. A regenerating clone of alfalfa (Medicago sativa) is Example 23 transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of Phenotypic Evaluation Procedure alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have 23.1 Evaluation Setup been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other com 0383. Approximately 35 independent TO rice transfor mercial alfalfa variety as described by Brown DCW and A mants are generated. The primary transformants are trans Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111 ferred from a tissue culture chamber to a greenhouse for 112). Alternatively, the RA3 variety (University of Wiscon growing and harvest of T1 seed. Events, of which the T1 sin) has been selected for use in tissue culture (Walker et al., progeny segregated 3:1 for presence/absence of the trans 1978 Am J Bot 65:654-659). Petiole explants are cocultivated gene, are retained. For each of these events, approximately 10 with an overnight culture of Agrobacterium tumefaciens T1 seedlings containing the transgene (hetero- and homo C58C1 pMP90 (McKersie at al., 1999 Plant Physiol 119: Zygotes) and approximately 10 T1 seedlings lacking the 839-847) or LBA4404 containing the expression vector. The transgene (nullizygotes) are selected by monitoring visual explants are cocultivated for 3d in the dark on SH induction marker expression. The transgenic plants and the correspond medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 ing nullizygotes are grown side-by-side at random positions. g/L K2SO4, and 100 um acetosyringinone. The explants are Greenhouse conditions are of shorts days (12 hours light), 28 washed in half-strength Murashige-Skoog medium (Murash C. in the light and 22°C. in the dark, and a relative humidity ige and Skoog. 1962) and plated on the same SH induction of 70%. Plants grown under non-stress conditions are watered medium without acetosyringinone but with a suitable selec at regular intervals to ensure that water and nutrients are not tion agent and suitable antibiotic to inhibit Agrobacterium limiting and to satisfy plant needs to complete growth and growth. After several weeks, somatic embryos are transferred development. to BOi2Y development medium containing no growth regu 0384 T1 events are further evaluated in the T2 generation lators, no antibiotics, and 50 g/L Sucrose. Somatic embryos following the same evaluation procedure as for the T1 gen are Subsequently germinated on half-strength Murashige-Sk eration but with more individuals per event. From the stage of oog medium. Rooted seedlings were transplanted into pots Sowing until the stage of maturity the plants are passed several and grown in a greenhouse. T1 seeds are produced from times through a digital imaging cabinet. At each time point plants that exhibit tolerance to the selection agent and that digital images (2048x1536 pixels, 16 million colours) are contain a single copy of the T-DNA insert. taken of each plant from at least 6 different angles. Cotton Transformation Drought Screen 0382 Cotton is transformed using Agrobacterium tumefa 0385 Plants from T2 seeds are grown in potting soil under ciens according to the method described in U.S. Pat. No. normal conditions until they approached the heading stage. 5,159,135. Cotton seeds are surface sterilised in 3% sodium They are then transferred to a “dry” section where irrigation is hypochlorite Solution during 20 minutes and washed in dis withheld. Humidity probes are inserted in randomly chosen tilled water with 500 g/ml cefotaxime. The seeds are then pots to monitor the soil water content (SWC). When SWC transferred to SH-medium with 50 g/mlbenomyl for germi goes below certain thresholds, the plants are automatically nation. Hypocotyls of 4 to 6 days old seedlings are removed, re-watered continuously until a normal level is reached again. cut into 0.5 cm pieces and are placed on 0.8% agar. An The plants are then re-transferred again to normal conditions. Agrobacterium Suspension (approx. 108 cells per ml, diluted The rest of the cultivation (plant maturation, seed harvest) is from an overnight culture transformed with the gene of inter the same as for plants not grown under abiotic stress condi est and Suitable selection markers) is used for inoculation of tions. Growth and yield parameters are recorded as detailed the hypocotyl explants. After 3 days at room temperature and for growth under normal conditions. lighting, the tissues are transferred to a solid medium (1.6 g/1 Geirite) with Murashige and Skoog salts with B5 vitamins Nitrogen Use Efficiency Screen (Gamborg et al., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/1 2.4-0, 0.1 mg/l 6-furfurylaminopurine and 750 ug/ml 0386 Rice plants from T2 seeds are grown in potting soil MgCL2, and with 50 to 100 ug/ml cefotaxime and 400-500 under normal conditions except for the nutrient solution. The ug/ml carbenicillin to kill residual bacteria. Individual cell pots are watered from transplantation to maturation with a lines are isolated after two to three months (with subcultures specific nutrient solution containing reduced N nitrogen (N) every four to six weeks) and are further cultivated on selective content, usually between 7 to 8 times less. The rest of the medium for tissue amplification (30°C., 16 hr photoperiod). cultivation (plant maturation, seed harvest) is the same as for Transformed tissues are subsequently further cultivated on plants not grown under abiotic stress. Growth and yield non-selective medium during 2 to 3 months to give rise to parameters are recorded as detailed for growth under normal Somatic embryos. Healthy looking embryos of at least 4 mm conditions. US 2015/0106974 A1 Apr. 16, 2015 49

Salt Stress Screen was converted to a physical Surface value expressed in square 0387 Plants are grown on a substrate made of coco fibers mm by calibration. The results described below are for plants and argex (3 to 1 ratio). A normal nutrient solution is used three weeks post-germination. during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt Seed-Related Parameter Measurements (NaCl) is added to the nutrient solution, until the plants are 0394 The mature primary panicles are harvested, harvested. Seed-related parameters are then measured. counted, bagged, barcode-labeled and then dried for three 23.2 Statistical Analysis: FTest days in an oven at 37°C. The panicles are then threshed and 0388 A two factor ANOVA (analysis of variants) is used all the seeds are collected and counted. The filled husks are as a statistical model for the overall evaluation of plant phe separated from the empty ones using an air-blowing device. notypic characteristics. An F test is carried out on all the The empty husks are discarded and the remaining fraction parameters measured of all the plants of all the events trans was counted again. The filled husks were weighed on an formed with the gene of the present invention. The F test is analytical balance. The number of filled seeds is determined carried out to check for an effect of the gene over all the by counting the number of filled husks that remained after the transformation events and to verify for an overall effect of the separation step. The total seed yield is measured by weighing gene, also known as a global gene effect. The threshold for all filled husks harvested from a plant. Total seed number per significance for a true global gene effect is set at a 5% prob plant is measured by counting the number of husks harvested ability level for the F test. A significant F test value points to from a plant. Thousand Kernel Weight (TKW) is extrapolated a gene effect, meaning that it is not only the mere presence or from the number offilled seeds counted and their total weight. position of the gene that is causing the differences in pheno The Harvest Index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground type. area (mm), multiplied by a factor 10°. The total number of 23.3 Parameters Measured flowers per panicle as defined in the present invention is the 0389 Biomass-Related Parameter Measurement ratio between the total number of seeds and the number of 0390 From the stage of sowing until the stage of maturity mature primary panicles. The seed fill rate as defined in the the plants are passed several times through a digital imaging present invention is the proportion (expressed as a %) of the cabinet. At each time point digital images (2048x1536 pixels, number of filled seeds over the total number of seeds (or 16 million colours) are taken of each plant from at least 6 florets). different angles. 0391 The plant aboveground area (or leafy biomass) is Examples 24 determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated Results of the Phenotypic Evaluation of the from the background. This value is averaged for the pictures Transgenic Plants taken on the same time point from the different angles and 0395 Transgenic rice plants expressing a nucleic acid was converted to a physical Surface value expressed in square comprising the Open Reading Frame of at least two genes mm by calibration. Experiments show that the aboveground encoding an iSYT polypeptide are evaluated under one or plant area measured this way correlates with the biomass of more of the conditions abovementioned (non-stress condi plant parts above ground. The above ground area is the area tions, drought stress, Nitrogene deficiency). The performance measured at the time point at which the plant had reached its of the transgenic plants outperform the control plants in one maximal leafy biomass. or more yield-related traits selected from aboveground bio 0392 The early vigour is the plant (seedling) aboveground mass (AreaMax), root biomass (RootMax and Root Thick area three weeks post-germination. Increase in root biomass Max), and for seed yield (total weight of seeds, number of is expressed as an increase in total root biomass (measured as filled seeds, fill rate, harvest index) and thousand kernel maximum biomass of roots observed during the lifespan of a weight in addition, plants expressing in addition, the trans plant); or as an increase in the root/shoot index (measured as genic plants comprising recombinant nucleic acids express the ratio between root mass and shoot mass in the period of ing at least two iSYT polypeptides or homologues thereof or active growth of root and shoot). fusions of the same show a faster growth rate (a shorter time 0393 Early vigour is determined by counting the total (in days) needed between Sowing and the day the plant number of pixels from aboveground plant parts discriminated reaches 90% of its final biomass (AreaCycle) and an earlier from the background. This value was averaged for the pic start of flowering (TimetoFlower: time (in days) between tures taken on the same time point from different angles and Sowing and the emergence of the first panicle).

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150106974A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3). US 2015/0106974 A1 Apr. 16, 2015 50

1. A method for enhancing a yield-related trait in a plant (ii) one or more control sequences capable of driving relative to a control plant, comprising increasing expression expression of the nucleic acid of (i); and optionally in a plant of: (iii) a transcription termination sequence. (i) any two or three nucleic acids encoding the correspond 12. The construct of claim 11, wherein one of the control ing two or three interactor of synovial sarcoma translo sequences is a plant promoter, a constitutive promoter, a cation (iSYT)-like polypeptides: GOS2 promoter, or a GOS2 promoter from rice. 13. A plant, plant part or plant cell comprising the construct (ii) two or three nucleic acids, each encoding a single of claim 11. iSYT-like polypeptide; or 14. A method for making a plant having increased yield, (iii) the nucleic acids of (i) and the nucleic acids of (ii), increased biomass, and/or increased seed yield relative to a wherein said iSYT-like polypeptide is selected from the control plant, comprising transforming a plant, plant part, or group consisting of any of the polypeptides of Table A, plant cell with the construct of claim 11. homologues thereof, and fusions of the same. 15. A method for the production of a transgenic plant 2. The method of claim 1 wherein at least one of the having increased yield, increased biomass, and/or increased polypeptides is a synovial sarcoma translocation (SYT) seed yield relative to a control plant, comprising: polypeptide or a homologue thereof, said SYT polypeptide or (i) introducing and expressing in a plant or plant cell any homologue thereof comprising an SNH domain having at two or three nucleic acids encoding the corresponding least 40% sequence identity to the SNH domain of SEQ ID polypeptides selected from the group consisting of any NO: 670. of the polypeptides of Table A or homologues thereof 3. The method of claim 1 wherein the nucleic acids encode and fusions of the same; and the corresponding polypeptides selected from the group con (ii) cultivating the plant or plant cell under conditions pro sisting of the polypeptides listed in Table 3. moting plant growth and development. 4. The method of claim 1, wherein the expression of the 16. A transgenic plant obtained by the method of claim 15, nucleic acids is increased by introducing and expressing the or a plant cell, plant part, seed, or progeny of said plant, nucleic acids in said plant. wherein said plant, or said plant cell, plant part, seed, or 5. The method of claim 1, wherein the nucleic acids are progeny, comprises any two or three nucleic acids encoding selected from the group consisting of the nucleic acids encod the corresponding two or three polypeptides selected from the ing any of the polypeptides listed in Table A and Tables A2 to group consisting of the polypeptides listed in Table A, homo Table A26, or is a portion of Such a nucleic acid, or a nucleic logues thereof, and fusions of the same. acid capable of hybridising with Such a nucleic acid. 17. A transgenic plant having increased yield, increased 6. The method of claim 1, comprising increasing expres biomass, and/or increased seed yield relative to a control sion in said plant of plant, resulting from increased expression of any two or three (i) a first nucleic acid encoding a polypeptide having at nucleic acids encoding the corresponding polypeptides least 80% sequence identity to the amino acid sequence selected from the group consisting of any of the polypeptides of SEQID NO: 2; and of Table A or homologues thereof and fusions of the same, or (ii) a second nucleic acid encoding a polypeptide having at a transgenic plant cell derived from said transgenic plant. least 80% sequence identity to the amino acid sequence 18. The transgenic plant of claim 17, wherein said plant is of SEQID NO: 120. a crop plant, a monocot, or a cereal, or wherein said plant is 7. The method of claim 1, wherein the enhanced yield rice, maize, wheat, barley, millet, rye, triticale, sorghum, related trait comprises increased yield, increased biomass emmer, spelt, Secale, einkom, teff, milo, or oats. and/or increased seed yield relative to a control plant. 19. Harvestable parts of the transgenic plant of claim 17, 8. The method of claim 1, wherein the enhanced yield wherein said harvestable parts are shoot biomass and/or related trait is obtained under non-stress conditions. seeds, and wherein said harvestable parts comprise any two or 9. The method of claim 1, wherein the enhanced yield three nucleic acids encoding the corresponding two or three related trait is obtained under conditions of drought stress, polypeptides selected from the group consisting of the salt stress or nitrogen deficiency. polypeptides listed in Table A, homologues thereof, and 10. The method of claim 4, wherein the nucleic acids are fusions of the same operably linked to a plant promoter, a constitutive promoter, 20. Products derived from the transgenic plant of claim 17 a GOS2 promoter, or a GOS2 promoter from rice. and/or from harvestable parts of said plant, wherein said 11. A construct comprising: products comprise any two or three nucleic acids encoding (i) any two or three nucleic acids encoding the correspond the corresponding two or three polypeptides selected from the ing two or three polypeptides selected from the group group consisting of the polypeptides listed in Table A, homo consisting of any of the polypeptides listed in of Table A logues thereof, and fusions of the same. or homologues thereof and fusions of the same; k k k k k